A Collection of Enzyme Assays

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CHAPTER 7A C ol lec t ion o f En zym e A ssay s

To measure an enzyme is in some ways easier, but in many waysharder than to measureametabolite. It is easier because an enzyme isa catalyst and can usually be made to generate a great deal more of itsproduct than is present in the tissue of origin. For example, musclelactate dehydrogenase can generate in an hour 100,000 times morelactate than is ever present in resting muscle. It is harder to measurean enzyme because, unlike m etabolites, which always have identicalproperties wherever they are found in the biological world, enzymeshaving the same function can differ greatly. Each enzyme that catalyzesaparticular reaction will more often than not differ from speciesto species, from cell type to cell type, and even from one kind oforganelle to another. The differences may concern kinetic properties,optimum pH, sensitivity to activators and inhibitors, and so forth. Inaddition, the activity of every enzyme is affected by tem perature ,pH ,and usuallyby ionicstrength,typeof buffer, andsoforth. Finally,an enzyme is inareal sense alive, and most enzymes can easily die orbe killed. All of this presents a challenge to the analyst, especiallywhen the protocol has not been designed for the particular cell typeunder examination.

E x p r e s s i o n o f E n z y m e A c t i v i t i e sThe International Unit (IU or simply U) is defined as the activitythat forms1|imolof product/min under specified conditions. Standardpractice is to express activity of isolated enzymes as U/mg of enzymeprotein. Tissue activities are more often expressed as mol, mmol, andso on/min or h/unit oftissuewetwt,drywt,or protein, at a given tem perature. Here we have usually expressed examples of tissue activitiesas mol (or mmol)/kg wet or dry wt at 20C. Another standard unit,rarely used in biological studies,is the katal or kat, whichisdefinedas the activity that will increase the rate of reaction by 1 mol/s.

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230 Passonneau and LowryTh e 44 methods presented are based on the decrease in N A DH or theformation of N A D +in 20 cases, the formation of N AD PH in seven teen

cases, the formation of NADH in seven cases, and the formation ofN A D P +in onecase.The op tions presented u sually call for change s inNA DH or NA DP H to be measured by changes in fluorescence or UVabsorption. Changes in N A D +are measured by the fluorescence pro duced with strong NaOH or in a few cases, by converting the NAD +back to NADH.V a r i a t i o n s i n A s s a y s f or A n y G i v e n E n z y m e

1. Directassay in thespectrophotometer i.e.,there isanincreaseor decreasein NADH or NADPH that can be followed directly in the instrument.This has an advantage in simplicity, but a disadvantage in that, whenmeasuring enzymes of low activity, the tissue homogenates cannot bediluted enough to avoid problems of turbidity and of disturbing sidereactions caused by other enzymespresent.The low dilution may makecontrol of effects of other tissue enzymes difficult.2. Directassayin thefluorometer.This eliminates some of the disadvantages with direct assays in the spectrophotometer. Because of the muchgreater sensitivity, tissue homogenates can usually be diluted toapointwhere turbidity is notaproblem;and because the reaction is carried outin inexpensive tubes, large numbers of samplescan beanalyzed simultaneously with repeated readings made over periods as long as an hour ormore. Thereis,however, one limitationtodirectfluorometricassays thatis illustrated by an example. TheKmfor NADH with glutamate dehydrogenase is 4\xM. In a spectrophotometer, the NADH concentration can bekept far enoughabove thislevel throughouttheassayto keep thevelocityat95%ormore of the V ^ . In thefluorometer,f the initialNADHconcentration is 10\xM(i.e., about the upper limit for fluorometer linearity),the initial rate will be only70%of V ^ and would fall to55%when theNADH falls to 5 \iM.However, this limitation for direct assays does notapply if the NADH oxidation is made by an auxiliary enzyme catalyzingasequential reaction. For example, in the directfluorometricassayfor aspartate aminotransferase,theoxaloacetate generatedby thetransaminase is rapidly converted to malate with NADH at an initial concentration of only10pM plus a relatively high level ofmalatedehydrogenase.Note that with both spectrophotometric andfluorometricdirect assays,if theNADHor NADPHisgeneratedorremovedbyan auxiliary enzymeor enzymes, there may be a significant initial lag until the auxiliaryenzyme(s) catchup.Therefore, it is important to take a series of readings to obtain linear (steady-state) reaction data seeChapter 2).


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Collection of Enzyme Assays 2313. Indirect assay in the fluorometer. The specific action is allowed to pro ceedfor afixed time (usuallyanhour), then stoppedand theproductmeasuredina variety ofwaysdependingonwhetherthefirst step involveda pyridine nucleotideorwhether the pyridine nucleotide reaction tookplacein theauxiliary second step. Ordinarily,theentire procedureiscarried outinthefin lf lu o r o m t rube.4. Indirect assay with enzymatic cycling. This differs from the above onlyin that the pyridine productisfinally amplifiedtowhatever degreeisnecessary. Usually, thefirststep iscarried out in a smaller vessel thanafluorometer tube: serological tubeoroil w ell.

Becauseitwas outofthequestion to provide protocolsforall circumstances,itis hoped that the analyst, with theaid ofthe many exam plesgiven, w ill easily adapttheproceduresto his or herparticular need s.A d e n y l a t e D e a m i n a s e (EC3.5.4.6) 1)

Thereare twoseparate enzym atic steps:1) A M P - > I M P+ NH/

[Re.7-1]2) NH4++a-ketoglutarate glutamate

H++NADH ^ * N A D+The auxiliary enzyme is glutamate dehydrogenase (GDH).Theactivity ismeasuredby the NADH decrease orN A D+ generatedinStep2.The deam inase is very unstable under assay cond itions, and thecond itions selected are a com prom isetoincrease stability, w hereas thereaction isinhibited by5 0% .The reagent chosen increasesthe half-lifeofthe enzym e from 10-15 min toabout175 min.

Sample tissue activities (mol/kgdrywt/h, 20): kidney, 0.6; rabbit soleusmuscle, 3 ; and rabbit tibialis anterior,20.Spectrophotometer

(0.3-1.5 nmol/min, 20-90 nmol of Product)Reagent1:Imidazole buffer, pH 6.5 (15 mAf imidazolebase,35mAf imidazole acetate);KC1,O.lAf;glycerol, 30% ; ATP, 1mAf;5'AMP,5 mM;dithiothreitol,1 mM; and bovine serum albumin, 0.05% .


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232 Passonneau and LowryReagent2:Tris-acetate buffer, pH 8.8 (85mAf Trisbase, 15 mAfTrisacetate); oc-ketoglutarate, 2 mAf; NADH, 0.1 mAf; ADP, 100 yJd\bovineserum albumin, 0.04%;andbeef liver glutamate dehydrogenase, 36 U/mL(300jig/mL)fromyophilizedpowder or inglycerolto avoidNH4+.

To further minimize ammonia contamination, the buffers are prepared just before use fromthebuffer bases, plus sufficient acetic anhydride to provide the acid equivalents. This procedure (devised byStephen H. H erm an , personal comm unication) converts any amm onia presenttoacetamide, and the acetic anhydride is converted w ithin10 min to acetic acid. The water, buffer base, glycerol, and KC1 arecombined, the acetic anyhydride added, and after 10 min, the othercomponentsareadded.Pureacetic anhydrideis9.5M, equivalentto19mol of acetic acid/L.

ProcedureStep 1: To 0.1mLofreagentin a10x 75mmtube is added an appropriateamount oftissueto be analyzed (e.g.,10pg of fast-twitch muscle, wetwt) or 10 pL of 3 -5 mAf (NH 4)2S04standard.Step2:Incubate at 25C for1 h.Step 3: Add 5 pL of0.7Af HC1;heat at 60 20 min to stop the enzymeactivity.Step 4: Add 1 mL of reagent 2, and read in spectrophotometer when thereaction is complete, approx 20 min.

Fluorometer Direct Assay(0.03-0.12 nm ol/min, 2-8 ntnol of Product)

Reagent1:The same as for Spectrophotometer.Reagent2:The same as for Spectrophotometer, except NADH, 10\iM.Procedure

Step 1: To 0.1 mL of reagent1is added the appropriate amount of tissue(equivalentto1-2\igof fast-twitchmuscle,wetwt).Standardsare 5\iLof 1.5 mAf (NH 4)2S04. Incubate at 25 for 60 min.Step2:Add5pL of0.7AfHC1;heat at 60 for 10 min.Step3:Add1 mLofreagent2.Read when reaction iscomplete,about 20min.Fluorom eter Indirect Assay

(3-15 pmol/min, 0.2-1 nmol of Product)Reagent1:Thesameasfor Spectrophotometer.Reagent2:Thesameasfor Spectrophotometer.


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Collection of Enzym e Assays 233Procedure

Step 1: One to 3 pL of tissue homogenate (e.g., 40 -200 ng of muscle wetwt)areadded to50pL of reagent 1. Standardsare60-120\iM(NH4)2S04added in the same volume. Incubate 60 min at room temperature.Step2:Add5\iLof0.7AfHC1;60 for 10 min.Step 3 : Add0.1mL reagent2;incubate 20 min at room temperature.Step4:Add 20 pL lAfHC1;room temperature for 10 min.Step5:Transfer 50 pL to1 mL of 6Af NaOH containing10mAf imidazole;heat 10 min at 60. Cool and read on the fluorometer.

A d e n y l a t e K i n a s e ( E C 2.7.4.3) (2,3)The four enzymatic reactions are combined. The AMP is removedto prevent product inhibition.

ADAAMP - IMP+ NH4+

2ADP-


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234 Passonneau and LowryProcedure

One m illiliter of reagent is added to cuvets, and a reading is taken.Tissue homogenateisadded to giveanOD change of0.030-0.060/min(5 -10 (ig of muscle, 80-150|agofbrain,wetwt).Readings are takenevery1 or 2 min.

Fluorometer(0.02-0.2 nm ol/min, 1-10 nm ol Product)Reagent1:The reagent is the same asfor Spectrophotometer, excepttheglu-cose-6-PdehydrogenaseandNADF*areomitted.Reagent 2: Tris-HCl buffer, pH 8.0 (25raMTris base, 25 mAf Tris HC1),NADP+, 50\sM\EDTA1 mM; glucose-6-P dehydrogenase, 0.28 U/mL0.5Mg/mL);and rabbit muscleP-glucoisomerase,0.5U/mL 1Mg/mL).

ProcedureStep1:Samples (e.g.,50 |xgkidney,0.1 jig muscle, wetwt)are added in avol of1pL into50\iLof reagent1in10x75 mmtubes.Standards are50 pL of0.1and 0.2mM ATPin reagent1,60 min at 25.Step2:Avol of5pL of lAfHC1is added to stop the reaction; 10 min atroom temperature.Step3:Add1mL of reagent2;10-15 min at room temperature. Read onfluorometer.

A l a n i n e T r a n s a m i n a s e ( E C 2.6.1.2) (1,4)1) Alanine +a-ketoglutarate>pyruvate + glutamate

2) Pyruvate + NADH - > lactate + NAD*The auxiliary enzyme is lactate dehydrogenase (LDH). The twoenzyme stepsarecarried out separately. This permits destruction afterenzyme Step1of potentially disturbing side reactions caused by NADHoxidase and glutamate dehydrogenase in tissue samples. Forthesamereason,a direct assay combining the two reactions above is impractical.Rather than measure the decrease in NADH in Step 2 above, theNADHisdestroyed withHC1,andtheNA D+isconvertedtoits highlyfluorescent strong alkali product. This extends the analytical rangealmost tenfold and makes it unnecessary to predict carefully the amountof NADH to use in Step2.W hen still more sensitivity is required, theNAD+is measured by enzymatic cycling.


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Collection of Enzym e Assays 235Sample tissue activities (mol/kg dry wt/h, 20): rat kidney, 0.03 -0.16; rabbit tibialis anterior muscle, 0.4; and rabbit soleus muscle, 1.2. Activities are about40%higher at 25.

Fluorom eter Indirect Assay(0.2-10 nm ol of Product)

Reagent 1: Tris-HCl buffer, pH 8.1 (25 mAf Tris base, 25 mAf Tris-H Cl);L-alanine, 50 mAf; monosodium a-ketoglutarate, 0.5 mM; pyridoxalphosphate, 5 \iM\and bovine serum albumin, 0.02% .Reagent2:Imidazole buffer, pH7(50 mAf imidazole-HQ, 50 mAf imidazole base),NADH,200\xM,andbeefheart lactatedehydrogenase,0.5U/mL 2^ig/mL).Standardsare5\\Lof 0.2-2 mAfpyruvatecarried throughouttheprocedure.

ProcedureStep 1: To 100\iLof reagent1 in10x75mm fluorometer tubes are added5 pL of an appropriate tissue dilution (10-5 0\igofkidney,2-25 jig ofsoleus muscle). Incubate1 h at 25, followed by 2 min at 100.Step 2: Add 100\\Lof reagent2;room temperature 15 min.Step3:Add 20\\L \MHC1;room temperature at least 15 min.Step4:Add1 mL 6Af NaOH containing 10mAfimidazole; 60 for 20 min.Cool and read on the fluorometer.

CommentT he Km for alanine is very high, about 50 mAf. This means theobserved activity with the recomm ended reagent is only about half theVmarIt also means that it is essential to test the purity of the L-alanine,since som e batches have been contam inated with L-aspartate or pyru

vate. Either contaminant would give erroneously high values.A l d o l a s e (5,6)( F r u c t o s e - 1 , 6 - b i s p h o s p h a t e , E C 4.1.2.13)

Th e three enzym atic steps are com bined (note that 2 mol of NA D +are generated/mol of fructose bisphosphate consumed):glyceraldehyde-P ^

Fructosebisphosphate ^ - s ^ \ TPI1 [Re.7-4]dihydroxyacetone-P 2 gtycero-P

2NADH *2 NAD+


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236 Passonneau and LowryThe auxiliary enzymes are triose-P isomerase (TPI) andglycero-Pdehydrogenase (GO P). (The spectrophotometry methodisnot recommendedforunfractionated tissueingeneral, becausetheamountoftissue requiredinmost cases would cause problems ow ing to turbidityand competing tissue enzym es.)Sample tissue activities (mol/kg dry wt/h,20):ratbrain, 1.2; mouse brain,0.6; rat kidney cortex, 0.4;andliver, 0.2.

Spectrophotometer5-10 nmollmin, 50-100 nmol of Product)

Reagent: Tris buffer, pH 7.7 (17mM Tris base,33mMTris-HCl);fructose-1,6-bisphosphate, 0.3mM; NAD H, 150|iM; bovine serumalbumin, 0.02% ; triose-P isomerase 10 U/mL; glycero-P dehydrogenase,0.7U/mL; and sodium amytal,2mM.Conduct of the Assay

The reaction is carriedout in 1 mLof reagentin acuvet.The sampleisadded,and the rateof oxidation of NA DH monitored for 10 or 20 m in.

Fluorometer Direct Assay0.1-1 nmol/min, 1-8 nmol of Product)

Reagent:Thereagentis thesameasfor Spectrophotometer, except NADH,10nM.Conduct of the Assay

The reaction is carried out in1mL ofreagent,following the disappearance of NADH. Appropriate tissue amounts are 50 -2 00|igof kidney,and 15-50|ig ofbrain, wet w t.

Fluorometer Indirect Assay2-100 pmol/min, 0.1-5 nmol of Product)

Reagent:Thereagent isthesameasfor Spectrophotometer, except NADH,1 mM; and nicotinamide, 20 mM.Procedure

Step1:To 50\\Lof reagentin 10x75 mmtubes areadded5\HLof sample containing, for example,10|ig of liver or brain. Standardsare 5 pLof 2mMNAD+ordihydroxyacetone-Pin thecompletereagent.Incubate60 min at25.Step2:Add0.5jiLof 5M HClat the same rate as thesampleaddition.Waitatleast 10 min.


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Collection of Enzym e Assays 237Step3 :Add1 mL of6Af NaOHcontaining10mAf imidazole; 30 min at 38or 10 min at60.Cool and read.

CommentTh e nicotinam ide prevents the enzym atic destruction of the productN A D + . Tissue blank s with the substrate omitted are needed becau se ofthe likelihood ofNADH oxidation by other tissue enzymes.y - A m i n o b u t y r a t e T r a n s a m i n a s e (E C 2.6.1.0) (7,8)

1) y-Aminobutyrate succinate semialdehydea-Ketoglutarate*** 'glutamate

2) Succinate semialdehyde succinate[Re. 7-5]SSDH

NADP+ ^ f NADPH + H+The auxiliary enzyme is succinate semialdehyde dehydrogenase(SSDH). The transaminase reaction is allowed to proceed in a firststep ; the oc-ketoglutarate remaining is destroy ed with H 2 0 2 . It is thenpossible to measure the succinate semialdehy de formed in the reactionwith commercial GABAse, in spite of the fact that it contains bothGA B A transam inase and succinate semialdehyde dehyd rogenase. Th e

pH for the measurement of succinate semialdehyde is shifted to 7.0beca use it is more favorable for the dehydrogen ase rea ction. At pH 7.0,th eKmfor succinate semialdehyd e is about2\iM, which m inimizes theamount of enzyme required.Sample tissue activities (mol/kg dry wt/h, 38): retina (neuronal layers),rabbit 0.02-0.16; monkey, 0.05 -0.33 ; and mouse brain, 0.5.

Spectrophotometer(0.5-2.5 nmol/min, 30-150 nmol Product)

Reagent: Tris-HCl buffer, pH 8.7 (85 mAf Tris base, 15 mAf Tris-HCl);GAB A,10mAf; oc-ketoglutarate, 5 mAf; pyridoxal phosphate, 100jiAf;mercaptoethanol, 1 mAf; and bovine serum albumin, 0.02% . (Theaddition of oc-ketoglutarate shifts the pH to 8.5.)


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238 Passonneau and LowryReagent2:Imidazole buffer, pH 6.5 (20 mAf imidazole base, 80 mAf imida-zole-HCl); NADP*, 500\iM;GABAse,0.025U/mL (25 ^g/mL); bovineliver catalase, 65 U/mL (1 fig/mL ); and bovine serum albumin, 0.02% .

ProcedureStep 1: Sam ples are added inavol of 5- 1 0 pL (equivalent to0.3-1.5mg ofbrain, wet wt) to 100 pL o f reagent1 in a 10 x 75 m m tube; incubate at38 for 60 min.Step 2 : Add 100 jiL of 20 mM H 2 0 2in0.05MNaOH; heat 30 m in at 38.This step destroys thea-ketoglutarateand tissue enzymes.Step 3: Add 1 mL of reagent 2, and transfer to spectrophotometer cuvet to

read when reaction is com plete, approx 2 0 m in.Fluorom eter Indirect Assay

(0.02-0.2 nmol/min, 1-10 nmol of Product)Reagent 1: The reagent is the same as for Spectrophotometer.Reagent 2: The reagent is the same as for Spectrophotometer, except theNADP+ is reduced to 100\xM; and the GABA se is omitted.

ProcedureStep 1: The samples (equivalent to 10-100 jig of brain tissue, wet wt) areadded in a vol of 5-10 \iLto 50 \iLof reagent in a 10 x 75 mm tube;incubate 60 m in at 38.Step 2: Add 50 ^L of 2 0 mM H 2 0 2in0.05MNaOH ; heat 30 min at 38.Step3:Add1 mL of reagent 2 ,andread at fluorometer setting equivalent to2 - 1 0 \iMNADPH at full scale deflection. Add 0.001 U/mL (1 >ig/mL)GABAse. (The comm ercial preparation usually has sufficient succinatesemialdehyde dehydrogenase to complete the reaction in 10 min.)Cycling Assay

(0.1-0.5 pmol/min, 5-25 pmol Product)Reagent: The reagent is the same as in Spectrophotometer.Reagent 2: The reagent is the same as in Spectrophotometer, except that

NADP+ is reduced to 10\iM.Procedure

Step 1: Freeze-dried samples (10 ng brain, 20 ng retina, dry wt) are addedto 0.2 pL of reagent 1 under oil. Standards are 0.2 jiL of 0.5-2 \iMsuccinate semialdehyde. Incubate 6 0 m in at 38.Step 2 : Add 0.2 ^L of 2 0 mAf H 2 0 2in0.05MNaOH, 30 m in at 38.Step 3: Add1\iLof reagent 2; 2 0 m in at room temperature.Step4:Add 1 ^L of 0.15 NaOH, 2 0 min at 80


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Collection of Enzyme Assays 239Step 5: Transfer 1 pL into a 10-75 mm tube containing 100 pL of NADPcycling reagent with enzyme levels to provide 1000-fold amplificationfor brain or 2000-fold amplification for retina in 60 min at 38.Step 6: Complete the indicator step as described in Chapter 5 .

CommentSuccinate semialdehy de recently has been made available com mer

cially.T he succinate semialdehyde dehyd rogenase activity in the m ixedGABAse preparation can therefore be tested for the content of thedehy drogen ase activity in advance of the tissue measu rem ent of transaminase activity.

A s p a r t a t e T r a n s a m i n a s e (E C2.6.1.1) (1,4,9)cx-ketoglutarate glutamate .R e 7

aspartate/^^ ^ôxaloacetate malateMDH

H++ NADH \ , NAD*The auxiliary enzyme is malate dehydrogenase (MDH). The twoenzyme steps can be combined because the activity is usually high eno ughso that nonspecific tissue oxidation of NA DH wo uld notbedisturbing.(This contrasts with the alanine transaminase assay, see first paragraph ofAssay for Alanine Transaminase.)

Sample tissue activities (mol/kg drywt/h,20):rat kidney cortex,25;mousebrain, 6; rabbit tibialis anterior muscle, 6; and rabbit soleus muscle, 8.Spectrophotometer(1-5 nm ollmin, 20-100 nm ol of Product)

Reagent: Tris buffer, pH 8.4 (35 mA/Tris base,15mMTris-HCl); monoso-dium oc-ketoglutarate,8mAf; monosodium aspartate 40 mAf; NADH, 100-150\xM;bovineserumalbumin, 0.02%;cytoplasmicmalate dehydrogenase,(pig heart) 1.2 U/mL 1îg/mL); and pyridoxal phosphate,10\iM.Conduct of the Assay

To 1 m L of re ag en t in a cuv et is ad ded 50 îL or less of hom o-genate co ntaining an appropriate am ount of tissue (e.g., 50 (ig of kidney or 2 00 (ig of skeletal m uscle w etwt).Blanks are reagent without


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240 Passonneau and Lowrya-ketoglutarateor aspartate, with tissue added. Readings are made at2-minintervals, andtherate calc ula ted fromthelinear portionofthe curve. Fluorometer Direct Assay

0.05-1 nmollmin, 1-10 nmol of Product)Reagent:Thereagent isthesameasfor Spectrophotometer, except NADH,5-10\iM.

Conduct of the AssayOne milliliter of reagent is added to10x75mm tubes. Readings aremade to give full-scale fluorescence. A n appropriate amountoftissueis added (5 -1 0|igkidney; 2 0- 50|igskeletal muscle or brain,all wetwt) and readings are taken every 2 or3min. The activityiscalculatedfrom thelinear portionofthe curve.

Fluorometer Indirect Assay2-20 pmol/min, 0.1-1 nmol of Product)

Reagent:Thereagent is the sameasfor Spectrophotometer, except NADH,5-10 M^.Procedure

Step1:To100 nLofreagent in a 10x75 mm tubeisadded theappropriateamountoftissue (0.5-1.5 |xg ofkidney,0.1-0.3|Xgofbrain in avolof 1-5^L). Standards are5-20\iMNAD+in100 ^L. Incubate60min at 25.Step2:Add 15 ^iL lAfHC1;room temperature 15 min.Step3:Add1 mL6AfNaOHcontaining10 mAfimidazole;60 for 20 min.Step4:Cool, dry,and readtubes on the fluorom eter.Cycling Assay

0.2-1 pmol/min, 10-50 pmol Product)The assay wasdeveloped especially for human ova, in w hich a0.05-|iLsample from 1 (iLofmedium added to one ovum was used.

Reagent:Thereagentwas thesameas forSpectrophotometer, excepta-ketoglutarate, 2 mM; NADH, 80\iM;andmalate dehydrogenase,0.6U/mL (0.5^ig/mL),with theammonium sulfateremoved bycentrifuga-tionandreplaced with reagent buffer.


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Collection of Enzyme Assays 241Procedure

Step 1: Approximately 0.05 \iLof tissue extract or, for example, 1 ng offreeze-dried brain is added to 2 pL of reagent under oil. Incubate1h at20.Standards are 0.05 iLof 0.8-2mMNAD+.Step2:Add 2XLof0.\MHC1;10 min at room temperature.Step 3 : Transfer 1p,L into a 10 x 75 mm tube containing 100\xLof NADcycling reagent with enzyme levels to provide 1000-fold amplificationin 60 min at 38.Step4:Complete the indicator step as described in Chapter 5 .

B r a n c h e d C h a i n A m i n o A c i d (1,10,11,12,13)A m i n o T r a n s f e r a s e ( E C 2 . 6 . 1 . 4 2 )

1) a-Keto-isocaproate leucine^ ^ ^r7

glutamate- 'a-ketoglutarate

[Re. 7-7]2) a-ketoglutarate + NH4* glutamate

NADH NAD+The auxiliary enzyme is glutamate dehydrogenase (GDH). The

specific reaction proceeds in the reverse of the usual direction. Theadvantageisthat the product measured (a-k etoglu tarate)ismuch lowerin tissue than is glutamate. The tissue blanks are thus lower, usually1-30% of the transaminase product.Sample tissue activities (mol/kg dry wt/h, 20): rat kidney, 0.04; rabbittibialis anterior muscle, 0.02; and rabbit soleus muscle, 0.03.

Fluorom eter Indirect Assay(2-30 pmollmin, 0.1-1 nm ol of Product)

Reagent: Imidazole buffer, pH 7.7 (85 m Mimidazole base, 15mMimida-zole-HCl); monosodium glutamate,25mM; oc-ketoisocaproate,120\iM;bovine serum albumin, 0.05%; and glycerol 20%.


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242 Passonneau and LowryReagent2 :Imidazole buffer, pH 8.6 (220 mAf imidazolebase,10mAf imidazole HC1); ammonium acetate, 50 mAf; NADH, 40 \iM\ADP, 200pAf; and beef liver glutamate dehydrogenase 1.2 U/mL (10 jig/mL).

ProcedureStep 1: To 100 pL of reagent 1 in 10 x 75 mm tubes are added 2-5 pL ofhomogenate (20-100\igof muscle or kidney, wet wt); 60 min at 25.Standards are 100 pL of 1-10\xMa-ketoglutarate.Step2:Add 15\iLof0.75MHC1;room temperature for 15 min.Step3:Add 100\ihreagent2;room temperature 15 min.Step4:Add 20 ^iL 2AfHC1;room temperature 15 min.Step5:Add1 mL 6Af NaOH containing10 mAfimidazole; 60 for 20 min.Cool, dry, and read tubes on the fluorometer.

CommentIt w as found, und er the analytical conditions used by Ichihara andKoyama 12) and by Taylor and Jenkins 13) (pyrophosphate buffer,pH 8.6), that over half of the enzy m e activity of m uscle hom og enateswa s lost in 60 m in. Ad dition of40% glycerol gave complete protec

tion, but activity was reduced b y almosthalf.A com promise w as to use20% gly cero l, which reduced initial activity< 5 %with a loss in 60 m inof only 10% . Th e buffer w as also changed to imidaz ole at pH 7.7,primarily to simplify the necessary acidification and neutralizationsteps. Ho wever, this also increased initial activity by 1 5 % . Th eM ichaelis constants under assay conditions were foundtobe13\iMfora-ketoisocaproateand 12 mAf for glutam ate. From this it may be calculated that observed activities are about 60% of the maxim al rate ofenzyme action (V m ax).

C a r n i t i n e A c e t y l t r a n s f e r a s e (E C 2.3.1.7) (1)The acetyl-CoA generated is converted to citrate in the first of twoenzyme sequences:1) acetylcarnitine carnitine

CoA acetyl-CoA CoA ^ ,CS

oxaloacetate citrate


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Collection of Enzym e Assays 243The auxiliary enzym e is citrate synthase (C S). Excess oxaloacetate isdestroyed with alkali and heat, and the citrate is measured by theN A D

+formed in the second enzym e sequence with citrate lyase (CL)and malate dehydrogenase (MDH):CLCitrate -* acetate + oxaloacetate malate

W H / [^. 7-9]NADH NAD+

Sample tissue activities (m ol/kg dry wt/h, 20): rabbit tibialis anterior muscle, 0.3 0, and rabbit soleus muscle, 0 .40.Fluorom eter Indirect Assay

(10-150 pmol/min, 0.5-10 nmol of Product)Reagent: Tris buffer, pH 8.1 (25 mAf Tris base, 25 mAf Tris HC1);acetylcarnitine, 5 mM; CoA, 2 mAf; oxaloacetic acid, 0.5 mM; citrate-free, Norit-treated bovine serum albumin, 0.02%; and pig heart citrate

synthase, 1.1 U/mL (10 îg/mL).Reagent2:Tris buffer 7.5 (20 mM Tris base, 80 mM Tris-HCl); NADH, 80\xM\ ZnCl2, 100\iM \bovine serum albumin, citrate-free, 0.01%; pigheart malate dehydrogenase, 1.2 U/mL (1 îg/mL); and Aerobacteraerogenescitrate lyase, 0.04 U/mL.Procedure

Step 1: To 50 iLof reagent1in 10 x 75 mm tubes are added 1-2 îL ofhomogenate (e.g., 10-100jxgof muscle, wetwt),25 for 60 min. Standards are 50 iLof 20-200\xMcitrate in reagent 1.Step2:Add5nL of 0.5MNaOH;95 for5 min.Step3:Add 0.5mLofreagent2;room temperature 20 min.Step4:Transfer 100\iLinto1 mL 6M NaOH with 10mMimidazole; 60for 20 min. Coolandread in the fluorometer.Comment

The oxaloacetate in reagent 1 is destroyed in Step 2. The citratelyasemay containcitrate, whichcan beremovedbywashingtheenzymepellet with 2 .5M (N H 4) 2 S0 4 . The assay is linear for 2 h. The volumesin Step1 areminimizedtoreduce citrate contamination.Thesamples arediluted tenfold in Step 3 to avoid inactivation of the citrate lyase bycitrate. The Michaelis constants of the rabbit muscle enzyme for CoAand acetylcarnitine are in the 0.05 and 0.2 mM range, respectively.


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244 Passonneau and LowryC i t r a t e S y n t h a s e ( E C 4 .1 .3 .7 ) (14,15,16)

The assay is conducted in two separate enzymatic steps.1) Acetyl CoA + oxaloacetate -* citrate + CoA

CL2) Citrate acetate + oxaloacetate malate _ _ _M D H ^ > [Re '7-1()J

H++ NADH NAD+The auxiliary enzymes are citrate lyase (CL) and malate dehydrogenase (M D H ). Th e substrate oxaloacetate of the first step is destroyedw ith hot alkali before enzy m e steptwo.This indirect proced ure allow sthe use of optimal substrate concentrations, and ends w ith a py ridinenucleotide step that permits amplif icat ion with cycling. Note theunusu ally high albumin level in the specific reagent. This is requiredfor maximum activity.

Th e results may b e erratic in the absence of preincubation, at leastw ith frozen-dried m uscle samples wh ich, unlike mo st tissues, do notdisintegrate in aqueous droplets. Other tissues should be tested todetermine if preincubation is necessary or helpful. Cycling Assayprovides an example of a preincubation procedure.Sample tissue activities (mol/kg dry wt/h,20):rat kidney cortex,4.6;rabbittibialis anterior muscle,2.7;rabbit soleus muscle,33;mousebrain,5.3;

dog myocardium, 17; and human vastus muscle, 3.8.Fluorom eter Indirect Assay(5-100 pmol/min, 0.3-6 pmol of Product)

Specific reagent: Tris-HCl buffer, pH 8.0 (25 mAf Tris base, 25 mAf Tris-HCl);acetyl CoA, 0.4mAf;oxaloacetate,0.5mAf;andcitrate-free bovineserum albumin, 0.25% . The oxaloacetate is added just before use froma lAf solution stored in0.5AfHC1 at -70 .Citratereagent:Tris-H buffer, pH 7.5 (10mAf Trisbase, 40 mAf Tris-HC l);ZnCl2,100 ^Af; NADH,30\iM\bovine serum albumin,0.01%(citrate-free);ascorbate, 4 mAf(fresh);citrate lyasefromAerobacter aerogenes,

0.006 U/mL; and pig heart malate dehyrogenase, 0.6 U/mL (0.5 |Xg/mL) with most ofthe(NH4)2S04removed by centrifugation.


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Collection of Enzym e Assays 245Procedure

Step1:Samples of 1-5pLof homogenateare added to50pLof the specificreagent, 60 minat25. Standards are 1-5 nmol of citrate, equivalent to1-5 \iLof1 mAfsolution.Step2:To all tubesadd 5^L of0.5AfNaOH;95 for5min.Step3:Add 500 jiL ofcitratereagentatleast 20 minat25.Step4:Add 30*iLof lAfHC1;20 min at 25.Step 5: Transfer 100- iL aliquot to 1 mL of 6Af NaOH containing 10 mAfimidazole; 20 min at 60. Cooland readin the fluorom eter.

Cycling Assay(0.03-O.30 pmol/min, 2-20 pmol of Product)

Preincubation reagent for muscle: 2-Amino-2-methyl-l ,3-pro-panediol, pH 8.8 (25 mAf 2 -am ino -2-m eth yl-l,3 - propanediolbase, 25 mAf 2-amino-2-methyl-l,3-propanediol-HCl); bovine serumalbumin, 0.25% (citrate-free); andKC1,0.6Af.(TheKC1is to dissolvethe muscle myosin.)Specific reagent: Tris-HCl buffer, pH 7.8 (17 mAf Tris base, 33 mAf Tris-HC1); otherwisethereagent is the sameasFluorometer Indirect Assay.Citrate reagent: Tris-HCl buffer, pH 7.0 (20 mAf Tris base, 180 mAf Tris-HCl); NADH, 50\iM; ZnCl2, 80 \UA\bovine serum albumin, 0.02%;citrate lyase from Aerobacter aerogenes,0.012 U/mL; and pig heartmalate dehydrogenase, 1.2 U/mL (1 ^ig/mL).Mai ate indicatorreagent:SeeChapter 5.

ProcedureStep 1:Eachtissue sample is introducedinto 1|iL of preincubation reagentinan oilwell;30-60min at25. This step isessentialfor musclesamplesbut may be omitted in the case of other tissues if tests show it to beunnecessary.)Step2:Add3\\Lof the specificreagent;20 for60min.Standards are3 iLof25and 50 |iAfcitratein the specific reagent.Step3:Add1^L of0.17AfNaOH;95 for 30 min.Step4:Cool andadd 5pL ofcitratereagent;20-25 for 20 min.Step 5: Add 2 \ihof0.5AfHC1;10 minat20-25.Step 6: Transfer 1 pL into 10 x 75 mm tubes containing 50 \iLof NADcycling reagent with enzyme levels to provide 1000-fold amplificationin1 h at 38.Step7:Complete the indicator step as described in Chapter 5.


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246 Passonneau and LowryCreatine Phosphokinase (EC 2.7.3.2) 3,14,17,18)

Th e three enzyme reactionsarecomb ined:Creatine-P .^Creatine

>ADP ATP ADPHK [Re.7-11]glucose ^ glucose-6-P 6-P-gluconolactone

G6PDHNADP+ NADPH+ H+

Th e auxiliary en zym esarehexokinase (HK ) and glucose-6-P dehyd rogenase (G6PDH ).Sample tissue activities (mol/kgdrywt/h, 20):rat single muscle fibers,

80-525;human vastus muscle,785;rat kidney, 0.18-18; rabbit tibialisanterior muscle, 1000; rabbit soleus muscle, 600; and m ouse brain, 50.Spectrophotometer

5-10 nmollmin 50-100 nmol of Product)Reagent: Imidazole-acetate buffer, pH 7 (65mAf imidazole base,35 mMimidazole-acetate);ADP,1mAf; creatine-P,25mAf; magnesium acetate,10 mAf; bovine serum albumin, 0.02% ; dithiothreitol, 5 mAf; AM P,20

mAfordiadenosine pentaphosphate, 10 nAf; glucose,2 mAf;NAD P\1.5mAf; hexokinase, 2.8 U/mL (20 jig/mL); and glucose-6-P dehydrogenase, baker's yeast, 0.18 U/mL (0.5 jig/mL).Conduct of the Assay

To1mL of reagent in a cuvet is added an appropriate d ilutionofthetissue to provide the amo untoftissue to be analyzed (skeletal m uscle,2-4 p,gwetwt; brain, 2 0- 40jig wetwt). Blanksarereagent withoutP-crea tine, but with tissue added. Readings are taken every 2 min. Therateiscalcu lated from thelinear portionofthe cu rve.

Fluorometer, Direct Method0.1-1 nmollmin, 1-10 nmol of Product)

Reagent: The reagent is the same as in Spectrophotometer, except NADP+,100MM.


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Collection of Enzyme Assays 247Conduct of the Assay

One m illiliter of reagent is added to 10 x 75 mL test tubes. A readingistaken with thefluorometersensitivity set at 10 -2 0divisions/ iAf.Tissuesamplesareadded(skeletalmuscle,0.05-0.1jig;brain, 0.5 -1\ig >mixed;and readings made every 2 min. The rate is calculated from the linearportion ofthecurve. Blanksare asfor Spectrophotometer. Standards are10 jiL of 0.5 and1 mAf AIP.

Fluorom eter Indirect Assay(0.02-0.2 nm ol/min, 1-10 nm ol Product)Reagent: The reagent is the same as for Spectrophotometer, except thatADP is omitted.

Conduct of the AssayStep1: The reagent is placed in 10-pL volumes in oil wells. Standards are10nLof0.1-10mAf ATP. Frozen-dried tissue samplesareintroducedinto the reagent through the oil.Step 2 : The reaction is started by the addition of 1pL of 10 mAf ADP at

measured time intervals.Step3:Thereaction is stopped 60 min after introducing the first sample bythe additionwith the same time intervalsof2\iLof100mAfEDTA thathas been adjusted to pH 10 with NaOH.Step4:Aliquots of10[iLare removed to 1mLof20mAf Tris-HCl,pH 8.Thebuffer fluorescence is read before and after the addition ofthesample.Fluorom eter Indirect Assay

(0.02-0.2 nm ollmin, 1-10 nmol of Product)This is a minor variation of the previous assay developed specifically for frozen-dried muscle sections that resist solubilization.Reagent 1: Imidazole-acetate buffer, pH 7.0 (60 mAf imidazole base, 40mAfimidazole-acetate);KC10.6Af;bovine serum albumin, 0.05%; anddithiothreitol, 0.5 mAf.Reagent 2: Imidazole-acetate buffer as in reagent 1; P-creatine, 25 mAf;

ADP, 1 mAf; MgCl2, 10 mAf; glucose, 3 mAf; bovine serum albumin,0.02%; dithiothreitol, 5 mAf; AMP, 20 mAf or diadenosinepentaphosphate, 10nAf;and yeast hexokinase 2.8 U/mL (20 jig/mL).Reagent 3: Tris-HCl buffer, pH 8.0 (25 mAf Tris base, 25 mAf Tris-HCl);NADP+,100\xM\EDTA,1mAf;baker'syeastglucose-6-P dehydrogenase,0.18 U/mL (0.5 ^ig/mL); and muscle P-glucoisomerase, 0.4 U/mL(1 ^g/mL).


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248 Passonneau and LowryConduct of the Assay

Step 1: Samples are introduced into1 \iLof reagent1 in oil wells for preincubation; 60 min at20.Standards are1 iLof 4-8mMATP.Step2:Reagent 2 is added in a vol of 10 pL ; 60 min at 20.Step 3 : The reaction is stopped by adding 2 fiL of 0.5M NaOH; 15 min atroom temperature.Step 4: An aliquot of 10\iLis removed to1 mL of reagent 3 . A reading ismade on the fluorometer when the reaction is complete, 10-20 min.

CommentsThe A M P is added to inhibit the adenylokinase present in the tissuesam ples. Presum ably the adenylokinase could be inhibited by the specific inhibitor, P^ -dK ad en osin e-S ^p en taph osp ha te, which is effectiveat much lower concentrations see GTP/GDP measurement) . However, it has no t been tested in this system . In the presen ce of AMP, theblank in samples incubated w ithout P-creatine is equiva lent to 1-2%of the creatine kinase activity of m uscle. In som e tissues, how ever, theactivity in the absence of P-creatine is an appreciable fraction of creatin eP-kinaseactivity and such blanks should alwaysbetested.Preincubationwas always included with frozen-dried samples to ensure activationby dithiothreitol and the solubilization of the dried muscle sections.

F r u c t o s e - 1 , 6 - B i s p h o s p h a t a s e (E C 3.1.3.11)(3,18,19,20,21)

The three enzyme reactions are combined:PG i [Re. 7-12]Fructose-l,6-P 2 > fructose-6-P - > glucose-6-P 6-P-gluconate

P, NADP+ NADPHThe auxiliary enzymes are P-glucoisomerase (PGI) and glucose-6-Pdehydrog enase (G6P DH ). The method describedisone used for m uscleand is based on the procedu re of Bass etal. 19).A somew hat modifiedreagent was used for kidney 20).Sample tissue activities (mol/kg dry wt/h20):rat kidney,3;rabbit kidney,1.25; rabbit tibialis anterior muscle, 0.40; rabbit soleus muscle, 0.04;rat single muscle fibers, 0.0003-0.40; and human vastus muscle, 0.38.

\


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Collection of Enzym e Assays 249Fluorometer Direct Assay

(0.1-0.5 nm ollmin, 5-10 nm ol of Product)Reagent: Imidazole-HCl buffer, pH 7 (30mAfimidazole base, 20mAfimi-dazole-HCl);EDTA,1mAf;bovine serumalbumin, 0.05%;p-mercapto-ethanol, 2mAf;MgCl2,2 mAf;NADP+,100 Af;fructose-1,6-P2,50\iM;rabbit muscle P-glucoisomerase, 0.25 U/mL (0.5 \ig/mL)\andbaker'syeast glucose-6-P dehydrogenase, 0.18 U/mL (0.5 jig/mL). Standardsare 10 pL of0.5and 1.0 mAf fructose-6-P .

Conduct of the AssayAreading is takenat afluorometer setting so that full-sca le fluores

cence is equivalentto3- 1 0 lAf NAD PH . Sam plesareadded inasmallvolume to give a rate of 2-5 divisions/min (0.05-0.5 (iAf). Approximately 10-5 0 |ig of kidney wet wt will give a satisfactory rate. Readingsaretaken and the activity calculated from the linear portion of thecurve. T issue blanksinreagent with fructose bisphosphate omitted areanalyzed simultaneously with the samples.

Fluorom eter Indirect Assay for M uscle(0.1-0.2 nm ol/min, 5-10 nm ol of Product)

Reagent: Imidazole-HCl buffer, pH 7.4 (45 mAf imidazole base, 20 mAfimidazoleHC1);fructose bisphosphate, 50\iM\ MgCl2, 40 mAf; KC1,0.3Af;ATP,10\iM;EDTA,1mAf;NAD+,50\xM\bovineserumalbumin,0.02%;rabbit muscleP-glucoisomerase, 2.5 U/mL 5pg/mL);andyeastglucose-6-P dehydrogenase, 0.5 U/mL (1.5 pg/mL).Procedure

Step 1: To1 mLof reagentin a 10x75 mmtube, add10\XLofanappropriate dilution ofthesample (40-100 fig of muscle). Standards are 10\\Lof0.5and1mAf fructose-6-P. Incubate 60 minatroom temperature.Step 2: Add 100 pL ofO.lAf Na2C03, mix and read on the fluorometerwithin 30 min (The shift inpHalmost completely stops the reaction.)Cycling Assay

(0.1-Q.25 pmollminlsample, 5-15 pmol of Product)Reagent 1: The reagent is the same as for Fluorometer Direct Assay (orFluorometer Indirect Assay for Muscle).

ProcedureStep 1: Addfreeze-driedsample to 5 pLof reagent1 inoil wells (e.g.,10 ngkidney cortex, 25 ng fast-twitch muscle, drywt).Standardsare 5pL of1-3[iMfructose-6-P (5-15 pmol). Incubate1 h at 20.


22/77

250 Passonneau and LowryStep 2: Add 1 pL of \M NaOH; heat at 95 for 10 min to destroy excessNADP*.Step3:Transfer 2-4 jiL into10x75 mmtubes containing100pL of NADPcycling reagent with enzyme levels to provide 2000-fold amplificationin1 h at 38.Step4:Complete the indicator step as described in Chapter 5.

KineticsThe apparentKmfor fructose bisphosphate is very low, 2\xMat pH 7an d 1 \LM at pH 8 (kidney ). H igher levels are used, although they aresomewhat inhibitory, to preserve linearity. NADP4 causes someinhibition at levels higher than 100\xM,perhaps owing to con taminationwith 5'AMP, which is known to be present in NADP* and to inhibitfructose bisphosphatase. The,for 5'AMP is approx 10\iM at pH 7and 30\xMat pH 8. Th e addition of p-mercap toethanol increased theactivity twofold. The reaction was linear for at least an hour. Thetemperature coefficient is8%/degreebetween 15 and 35 C.

Fumarase (EC 4 .2 .1 .2 ) (14,22)Th e analytical sequenc e is carried out in two steps to avoid oxidation of NA DH by tissue constituents. The m alate dehydrogenase (MD H)reaction is pulled to completion with aspartate transaminase (AT).1) Fumarate > malate

a-ketoglutarateglutamate

2) Malate oxaloacetate aspartate [ R e. 7-13]MDH

NAD+ NADH + H+Sample tissue activities (mol/kg dry wt/h 20): rat kidney cortex, 49; ratmuscle, 5 -22; human muscle,1.9-4.8;and rabbit brain, 6.


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Collection of Enzyme Assays 251

Spectrophotometer1-2.5 nmol/min, 60-150 nmol of Product)

Reagent: Potassium phosphate buffer, pH 7.0 (30 mAfK2H P 0 4 , 20 mAfKH2PO4); disodiumfiimarate,50mAf;and bovine plasma albumin, 0.05%.Reagent 2:2 -Am ino-2-m ethyl-l-propa nol buffer, pH 9.6 (15 mAf 2-amino-2-methyl-l-propanol base, 35mM2-amino-2methyl-1-propanolHC1);N A D + ,400 \iM\glutamate, 10 mAf; malate dehydrogenase,6U/mL(5^ig/mL);andglutamate-oxaloacetate transaminase, 0.4 U/mL (2 jig/mL).Procedure

Step1:T o 0.1 mL of reagent1in10x75 mm tubesisadded the appropriateamount of tissue (10 igof kidney wet wt, 100 igof skeletal muscle wetwt), incubate 1h at20; heat2m inat100.Step2:Add1 mL of reagent 2 (without enzym es), and read at 340nm .Addthe enzym es inavol of 5-1 0^iL.Read when reaction is comp lete, about15 min.

Fluorometer Indirect Assay0.05-0.15 nmol/min, 3-10 nmol of Product)

Reagent: The sameas forSpectrophotometer.Reagent2:The sam e as for Spectrophotometer, except the buffer is reducedto 20mAf, N A D+to50 lAf, and glutam ate to3 mAf.Procedure

Step 1: Add samples (e.g., 1\igofkidney,wetwt)to100 pLofreagent1.Incubateatroom temperature 60 m in; heat 2 min at 100. Standards are5\\Lof0.8 and2mAf malate added reagent1.Step2: Add 1 mL of reagent 2 (with enzymes). Read when reaction iscomplete, about15 min.

Cycling Assay1-3 pmol/min, 50-150 pmol of Product)

Reagent: The reagentisthe sam e asforSpectrophotometer.Reagent2:The reagentisthe sameasfor Spectrophotometer (with enzymes),except N A D + , 100nAf.Procedure

Step1: To 1 jiL ofreagentin oilwells are added freeze-dried samplesofappropriate size(20 ngmuscle dry wt). Standardsare1pL of 50 and150\ iM malate in reagent1.Incubate60min at 20 .


24/77

252 Passonneau and LowryStep2:Add1 pLof0.05MNaOH; heat20min at 80.Step3:Aftercooling to roomtemperature,add 5 pLof reagent2;20 min atroom temperature.Step4:Add5jiL of0.2AfNaOH; 20 min at 80.Step 5: Transfer 2\iLinto a10x 75 tube containing 100 p,L of NADcycling reagent with enzyme levels to provide 500-fold amplificationin1 h at 38.Step6:Complete the indicator stepasdescribed in Chapter 5.

G luc o se - 6 - P ho sp ha t a se (E C 3 .1 .3 .9 ) (20)The assay is conducted in two separate enzyme steps:

1) GIucose-6-P _ > glucose + P,2) Glucose glucose-6-P

ATP ADP ATP\ P K ^ | [Re. 7-14]P-pyruvate ^ pyruvate lactate

NADH NAD+The auxiliary enzymes are hexokinase (HK ), pyruvate kinase (PK),and lactate dehydrogenase (LDH). There are various ways describedto measure glucose-6-phosphatase, but glucose release has proven tobe the most satisfactory, particularly for small samples. The alternative of measuring?{liberationgives higher and more erratic tissueblanks.The glucoseisconverted backtoglucose-6-P with hexokinaseand ATP, and the ADP formed is measured by the enzyme pathwaygiven above.

Tissue Preparation for Larger Sam plesTissue is homogenized in50mM imidazole-HCl buffer, pH7.1 (33mAf imidazole base,17 mMimidazole HCl) containing1mMEDTAand 0.02% bovine serum albumin. Bovine serum albumin greatlyincreases the stability of the enzyme in the homogenate.

Sample tissue activity (mol/kg dry wt/h,20):rat kidney cortex, 2.0.


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Collection of Enzyme Assays 253Spectrophotometer(0.3-2 nm ol/min, 20-100 nm ol of Product)

Reagent 1: Imidazole-HCl buffer, pH 6.8 (50 mAf imidazole base, 5 0 mAfimidazole-HCl); EDTA, 1 mAf; bovine plasma albumin, 0.02%; andgluc ose-6 -P 10 mAf.Reagent 2: Imidazole-HCl buffer, pH 7.2 (35 mAf imidazole base, 15 mAfimidazole-HCl); KC1, 75 mAf; ATP, 100 \iM; P-pyruvate, 300jiAf;M g C l2 ,2 mAf; p-mercaptoethanol, 2 mAf; NA DH , 10 0- 15 0\xM;rabbitmuscle pyruvate kinase, 0.3 U /mL (2 jig/mL); beef heart lactate dehydrogenase, 6.25 U/mL (2.5 ^ig/mL); and yeast hexokinase, 0.3 U/mL

(2 \ig/mL) added after an initial reading.ProcedureStep 1: To 100 pL of reagent 1in 10 x 75 mm tubes are added 1-10 jiL oftissue homogenate containing the appropriate amount of activity (e.g.,5 0 - 2 0 0/|Ligw et wt of kidney), 60 min at 25 ; 2 min at 95 -1 00 . Blanksare reagent without glucose-6-P, but with tissue added.Step 2: Add 1 mL of reagent 2 (without hexokinase) and mix. Transfer tocuve ts, and read at 340 nm .Step3:Add 0.3 U of hexokinase in 2 -1 0 pL , mix and read when reaction isover, 10-15 min.

Fluorom eter Indirect Assay(0.02-0.2 nmollmin, 1-10 nm ol of Product)Reagent 1:The sam e as in Spectrophotometer.Reagent2:The same as inSpectrophotometer,except:ATP, 50\iM\P-pyruvate,30\iM ; NADH, 2-1 0\iM;andpyruvate kinase, 0.6 U/mL (5 ^ig/mL).

ProcedureStep 1:T o 100 of reagent1 add 10 pL of tissue homogenate containing, forexample, 2 -2 0 \igwet w t of kidney. Incubate 60 m in at 25 ; heat 2 m inat 95 -1 00. Blanks are as for Spectrophotometer. Standards contain 2 -10 nmol glucose.Step 2: Add 1 mL of reagent 2 without hexokinase, mix, and read at full-scale deflection on the fluorometer for the NA DH leve l used.Step 3: Add 0.3 U hexokinase in 2-10 jiL, mix, and read when reaction isover, 10-15 min.

Cycling Assay(0.15-1.5 pmol/min, 10-100 pmol of Product)Reagent 1: Same as for Spectrophotometer, except bovine serum albumin,0.04%.


26/77

254 Passonneau and LowryReagent 2: The reagent contains double the concentration of all components inFluorometer IndirectAssay,except:P-pyruvate,40\iM;NADH,50\sM\ and P-mercaptoethanol, 2 mAf. Before use, the enzymes arecentrifuged and the supernatant ammonium sulfate fluid removed. Allenzymes are included in the reagent.

ProcedureStep1:Reagent in volumes of1 \iLareintroduced underoilin the oil wells.Standards are1 \ihof 10-100\iMglucosein complete reagent. Freeze-dried samples 5-50 ng of kidney (dry wt) are added to the reagentthrough the oil. Incubate 60 min20;and then heat 20 min at 60.Step2:Add1 pL of reagent2 ;30 min 22-25.Step 3: Add 5 \\Lof 50 mMHC1 to stop the reaction and destroy excessNADH.Step 4: Transfer 1 jiL into a 10 x 75 mm tube containing 100 ^L of NADcycling reagent with enzyme levels to provide 500-fold amplificationin1h at 38.Step 5 : Complete the indicator step as described in Chapter 5 .

General CommentReagen t blanks can be troublesome. Many batches of glucose-6-Pcontain g lucose . Th is can be removed w ith glucose o xidase, but it maybe simpler to find a batch low in glucose. Acidification followed byheating after the first incubation increases the blank. Therefore, heatalone is used to stop the reaction. O ther contributions to the blank arethe result of slight contamination with

1. Pyruvate in the P-enolpyruvate (removable by heating for a few minutesat 100C with a slight molar excess of H 20 2);2.ADP in the ATP (removable by conversion to ATP,seecreatine assay,Chapter 6 ); and3.N AD+in the NADH (removed by heating just before use in weak alkali[pH 10-12]). The last is only important for Cycling Assay.G l u c o s e - 6 - P h o s p h a t e D e h y d r o g e n a s e

(E C 1.1.1.49) (16,23)glucose-6-P ^6-P-gIuconolactone

^ > - < ^ [Re. 7-15]NADP+ 2d NADPH + H+


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Collection of Enzym e Assays 255The activity is measured by the rate of formation of NADPH asshown. Modifications are described that permit the measurement of

enzym e activityin50ngof dry tissue. (The spectrophotometric methodis not recommended for unfractionated tissue in general, because theamount of tissue required in most cases w ould cause problems owingto turbidity and competing tissue enzym es.)Sampletissue activities mol/kg drywt/h, 20): human brain,0.40;mouse brain,0.60;ratliver,0.60;and ratkidney,0.50.

Spectrophotometer(2-10 nm ollmin, 50-150 nm ol Product)Reagent: 2-Amino-2-methyl-l,3-propandiol-HCl buffer, pH 9.4 (70 mAf2-amino-2-methyl-l,3-propandiol base,30 mAf2-amino-2-methyl-l,3-propandiol HC1); glucose-6-P, 2 mAf;NADP+,0.5mAf;EDTA,0.5 mAf;andbovine serum albumin, 0.02%.

Conduct of the AssayThe sample is added to1 mL of reagent in a cuvet (1 -5 mg). Read

ings are taken every 2 min, and the rate calculated from the linearportion of the curve.Fluorometer Direct Assay(0.02-1 nm ollmin, 1-10 nm ol of Product)

Reagent:The reagent is the same asfor Spectrophotometer,except:NADP+,50\\M,and glucose-6-P,1 mAf.Conduct of the Assay

One-milliliter volum es of reagent are added to 10 x 75 mm tubes.The sensitivity of the fluorometer is set for full-scale fluorescenceequivalent to 10 |iAf NAD PH . After an initial reading, an appropriateamount of tissue homogenate is added (15-500 p,g, wet wt, of brain,muscle, or liver). Readings are made at2-minintervals, and the ratecalculated from the linear portion of the curve. Tubes containing reagent without glucose-6-P,but withadded tissue serveasblanks. Standards are 1-10 nmol of glucose-6-P.

Cycling Assay(1.5-3 pmollmin, 5-25 pmol of Product)Reagent:The reagent is the same asfor Spectrophotometer,except:N ADP+,100\iM,and glucose-6-P,1 mAf.


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256 Passonneau and LowryProcedureStep1:To 5\xLof reagentin oil wells are added 10-50 ng dry wt) samples of

brain (for example). Standards are 5 pL of reagent containing 1-5 \iMNADPH. Incubate60min at 20.Step2:Add5^L ofO.lAfNaOH; 20 min at 80.Step3:Transfer2pL into a10x75 mmtube containing100pL ofNADPcycling reagent withenzyme levels toprovide 2000-fold amplificationin1 h at 38.Step4:Complete the indicator stepasdescribed in Chapter 5.Comment

The composition of the bufferischosen to give near maximal velocity.Becausethe nonenzymatic hydrolysis of6-P-gluconolactoneoccursmore rapidly at alkalinepH ,the high pH serves to minimize the effectof the reaction nearing equilibrium.Glutamate Decarboxylase (EC 4.1.1.15) (8)

1) Glutamate - y-aminobutyrate (GABA) +C022) a-ketoglutarate glutamate

^JABAT^, [ R e 7 1 6 ]GABA succinate semialdehyde succinate

\ S S A D H /

NADP NADPH + H+The auxiliary enzymesareGABA transaminase (GABAT) and succinatesemialdehyde dehydrogenase (SSADH),which are both presentin comm ercial GA BAse. The reaction is conducted in two steps,because the amount of auxiliary enzymes needed for a coupled assay

would contribute disturbing blankvalues.(The spectrophotometric methodis not recommended for unfractionated tissue in general, because theamount of tissue required in most cases would cause problems owingto turbidity and competing tissue enzymes.)Sample tissue activities (mol/kg dry wt/h, 20): rabbit retina, 0.05, andmonkey retina, 0.072.


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Collection of Enzyme Assays 257

Spectrophotometer0.5-2 nmol/min, 30-100 nmol of Product)

Reagent1:Imidazole buffer, pH 7.0 (30 mAf imidazole base, 2 0 mAf imidazo le HC1); pyridoxal phosphate, 160 jxAf;glutamate 10mAf;andbovineplasma albumin, 0.05%.Reagent2:Potassium pyrophosphate buffer, pH 8.76 (33 mAfK3HP207,67mAf K4P2O7); ot-ketoglutarate,1mAf; GA BA se, 0.4 U/mL (40 0 pg/mL );NADP* ,1mAf;dithiothreitol,1mAf; and bovine plasma albumin, 0.02%.(W e are indebted to D iane Durham for the information that additionofdithiothreitol increases activity and decreases variability.)

ProcedureStep 1: Samples are added in 10\\Lorless to 100 pL of reagent 1. Standardsare 10 pLof5- 10 mAf GAB A in comp lete reagent. Incubate1hat 38.Step 2 :Add10 pLof 1MHC1;10 min at 60 .Step 3:Add1 mLofreagent 2; incubate60min at room temperature,andread in cuvetsinthe spectrophotometer.

Fluorometer Indirect Assay0.03-0.2 nmol/min, 1.8-10 nmol of Product)

Reagent 1: The reagentisthe same asinSpectrophotometer.Reagent2:Thereagent is the sameasin Spectrophotometer, except: NA DP*,

50\\M.Procedure

Step 1: To 100 pL ofreagent 1 in 10 x 75mm tubesareadded 10\xLoftissue (0.2-1 mg ofbrain,wetwt),or 10 jiL of0.5 and 1mAf GA BAstandard. Incubate60minat 37.Step 2:Add10 pLof lAfHC1;10 min at 60 .Step 3 :Add1 mLofreagent 2; incubate60min at room temperature,andread on the fluorometer.

Cycling Assay0.005-O.02 pmollmin, 0.3-1.2 pmol of Product)

Reagent 1: The reagentisthe same asinSpectrophotometer.Reagent2:Thereagent is the sameasin Spectrophotometer, except: NADP+,25 MAf.

ProcedureStep1:Samples (e.g.,5-20 ngfreeze-dried brain)areaddedto 0.1\ihofreagent1 in oil w ells . Standards are 0.1 pLof10\iMG AB A in reagent1. Incubate 1hat38.


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258 Passonneau and LowryStep2:Add0.1jiL of75 mMHC1,andheat20min, 60.Step3:Add0.5jiLof reagent2,andincubate60 min at roomtemperature.Step4:Add1 jiL of0.2AfNaOH,andheat20min at 80.Step5:Transfer1 jiLintoa10x75 mm tubecontaining100\iLofNADPcycling reagentwith enzymelevels to provide15,000-fold amplificationin1h at 38.Step6:Complete the indicator stepasdescribed in Chapter 5.

CommentTissue blanks consist of tissue samples carried through the wholeprocedure with glutamate omitted from reagent 1. This is essential

because preformed GABA,at least inbrain,is present at a concentrationthat is a substantial fraction of the GAB A generated.G l u t a m a t e D e h y d r o g e n a s e ( E C 1.4.1.3) (1,9,24,25,26)

NH / +a-ketoglutarate glutamate> < [Re. 7-17]H++ NADPH NADFTheactivity of glutamate dehydrogenase canbemeasured with eitherNAD orNADP ascoenzyme, and itisfeasible to measure the activityeither in the direction of oxidation or reduction ofthecoenzyme. Thechoice of pyridine nucleotide and direction of the assay may be influenced by the presence of disturbing side reactions in tissues. In brainand muscle tissue, at least, there is NADH-oxidizing activity, whichwould create high tissueblanks.For this reason, a method with NADPH

as cofactorisdescribed.In muscle,theactivity with NADHwasfoundto be 8% higher than with NADPH. (The spectrophotometric methodis not recommended for unfractionated tissue in general, because theamount oftissuerequired in most cases would cause problems owingto turbidity and competing tissue enzymes.)Sampletissue activities mol/kg drywt/h,20):rabbit tibialisanteriormuscle,0.2; rat soleus muscle, 0.40; and rabbit brain, 0.45.

Spectrophotometer(5-10 nm ollmin, 25-150 nm ol of Product)Reagent1:Imidazole buffer,pH7.4 35mM imidazolebase,15mAfimidazole-HCl); a-ketoglutarate, monosodiumsalt,2mM; ammonium


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Collection of Enzyme Assays 259acetate, 25 mAf; ADP, 100 |iAf; bovine serum album in, 0.0 5 % ; andNADPH,200^ iM.

Conduct of the AssayAreading is m ade on the spectrophotom eter w ith 1 mL of reagent.Sam ples are added and readings taken every1or 2 min. Blanks containsamples added to cuvets containing reagent without a-ketoglutarate.Calculations are based on the linear portion of the assay.

Fluorom eter Indirect Assay(15-50 pmol/min, 1-3 ntnol of Product)Reagent 1: The reagent is the same as for Spectrophotometer, except thatNADPHislOOpM.Reagent2:N aOH, 6Af containing 10 mAf imidazole base.

ProcedureStep 1: To 100p,Lof reagent1 in10x75mm tubes, add 1-2 iLof muscleor brain diluted to contain 20 -40 jxgof tissue;1h at20.Standards are

l -3nmolofNADP+.Step 2: Add 10\iLof \MHC1;10 min at room temperature.Step 3 : Add1 mL of reagent 2; heat at 60 20 min. Cool and read.Cycling Assay(0.05-O.1 pmol/min, 3-6 pmol of Product)

Th e following assay w as developed for analyses of enzy m e activities in human ova.Reagent:Thereagentis the same asfor Spectrophotometer, except that 50\xMNADH is used (rather than NADPH).

ProcedureStep 1: To 2 ^iL of reagent in oil wells is added 0.1 pL of sample or standards of 25 -50 \iMNAD+.Step2:Add 2\iLofO.lAfHC1;10 min at room temperature.Step 3 : Transfer 2\iLinto a 10 x 75 mm tube containing 100 ^iL of NADcycling reagent with enzyme levels to provide 5000-fold amplificationin1 h at 38.Step4:Complete the indicator step as described in Chapter 5.


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260 Passonneau and LowryG l u t a m i n a s e (E C 3 .5 .1 .2 ) (27,28)

1) Glutamine + H20 -> glutamate + NH4+2) Glutamate a-ketoglutarate + NH4+

[Re.7-18]

NAD+ NADHTh e auxiliary enzym e is glutamate dehyrogenase (G DH ). The p rocedu re consists oftwosteps, because ow ing to the low catalytic a ctivity of glutamate dehydrogenase in the direction required, it is notpractical to add sufficient enzyme to provide a coupled assay.Th ere are two distinct isozy m es; one is P rde pe nd en t, and the otheris indepen dent ofPi9but activated 15-fold by maleate. Procedures forboth forms are given so that the relative content of any tissue can beassessed. The P rindependent isozyme has been found by Curthoysand K uhlenschmidt 28) to be y-glutamyltranspeptidase.

Sample tissue activities (mol/kg dry wt/h, 20): rat kidney, 1.3; and mousebrain, 0.8.Spectrophotometer

(2-4 nmol/min, 25-100 nmol of Product)Reagent 1: (Phosphate-dependent form) Tris-HCl buffer, pH 8.6 (40 mAf

Tris base, 10 mAf Tris-HCl); glutamine 20 mAf; phosphate pH 8 (140mAf K 2HP


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Collection of Enzyme Assays 261Step2:Add 15 jiL of 2AfHC1;10 minatroom temperature.Step 3: Add 1 mL of reagent 2, and read in the spectrophotometer after20-30 minatroom temperature.

Fluorom eter Indirect Assay(0.03-0.3 nm ollmin 1-10 nmol of Product)

Reagents 1 and 1 A: The same as for Spectrophotometer.Reagent2:The sameasfor Spectrophotometer, exceptthebuffer is 50mM(35 mM Trisbase, 15mM Tris-HCl) andthe NAD+is 300\iM.Procedure

Step 1: Reagent1or 1A is added inavol of20pL toa 5x 60mmtube.Step2:Tissuehomogenate samplesare added in avol of 1-3^Lcontaining,for example, 10 |xg of kidney or brain. Standards are 3 ^L of 1and 2mMglutamate. Incubate 60 min at 37.Step3:Add 3 pL of2MHC1;10 minat roomtemperature.Step4:Transfer 20jiL to 1 mLof reagent2.After 20min at roomtemperature, the fluorescence is measured.CommentThe peroxide in reagent2is included to destroy thea-ketoglutarateand ensure the completion of the dehydrogenase reaction. The AD P isincluded to stabilize the glutamate dehydrogenase.

Cycling Assay(0.4-4 pmol/min, 25-100 pmol of Product)

Reagents1 and 1A: The same as for Spectrophotometer.Reagent2:The same as for Fluorometer Indirect Assay.

Conduct of the AssayThe assay is conducted in oil wells.

Step 1: Freeze-dried samples (20-100 ng) are introduced into 0.25 \iLofreagent 1 or 1A in oil wells. Standards are 0.25 ^iL of 0.2-0.4mMglutamate in complete reagent. Incubate1 h at 20.Step2:Add 0.25 ^L of 0.3MHC1;10 minatroom temperature.Step3:Add5pL ofreagent2;60 min at 20.Step4:Add 2^Lof0.25MNaOH;20 min at 80.Step 5: Transfer 1 jiL into 10 x 75 mm tubes containing 100 iLof NADcycling reagent with enzyme levels to provide 500-fold amplificationin1 h at 38.Step 6: Complete the assay as described in Chapter 5.


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262 Passonneau and LowryG l y c e r o - 3 - P h o s p h a t e D e h y d r o g e n a s e(E C 1.1.1.8) (5,6,14,16)

Dihydroxyacetone-P elycero-P^ r - J ^ [Re. 7-19]H++ NADH NAD+The reaction is followed by measuring the rate of disappearance ofNADH or by measuring the strong alkali-induced fluorescence of theNAD+formed aftertheacid destruction of NADH. Further sensitivity

is achieved by cycling the NAD+product.Sample tissue activities (mol/kg dry wt/h,20):rat kidney,5;rat liver, 15;mousebrain,0.4;humanbrain, 0.27;rat tibialisanterior muscle,6;andsoleus muscle, 0.5.

Spectrophotometer(2-10 nm ollmin, 20-80 nm ol of Product)Reagent: Imidazole buffer, pH 7.0 (30mAfimidazolebase,20mAfimida-

zole-HCl); dihydroxyacetone-P,1mAf; NADH100\iM\bovine serumalbumin, 0.02%; and sodium amytal,2mAf.Conduct of the Assay

One-milliliter volumes of reagent are added to cuvets and initialreadings made. Tissue homogenate samples are added (50 |iL or lesscontaining in the order of 200 igfast-twitch muscleorkidney, or 100|lg of liver, all wetwts).Readings are made at2-minintervals. Blanksconsist of tissue addedtoreagent without dihydroxyacetone-P. Calculations are made from the linear portion of the curve.

CommentThe sodium amytal is addedtominimize the oxidation of NADH inthe absence of dihydroxyacetone-P. Fresh homogenates have moreamytal-sensitive, NADH-oxidizing activity than frozen or freeze-driedtissues. In the case of brain atleast,the freeze-dry ing process eliminates

the troublesome activity almost completely.Fluorometer(0.1-0.5 nm ollmin, 2-8 nm ol of Product)

Reagent:Thereagentis the same asSpectrophotometer, except NADH,lOpAf.


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One-milliliter volum es of reagentarepipetedinto1 0 x 7 5mmtubes.Initial readingsaremade sothat theNADH g ives full-scale deflectionon thefluorometer. Tissue is addedin a5-10-|iLvol containing 2 00[igslow -twitch or 2 0 -5 0 |J,g fast-twitch muscle, 25 |Lig kidney , 200 |Xgbrain, or 5 jutg of liver, all wet wts. Readings are taken at 1-5 minintervals. Tissue blanks consist of tissue added to reagent withoutdihydroxyacetone-P (not needed for high-activity tissues).

CommentTheKmfor NADH is 0.3 |iM. Therefore, the average rate over thetotal interval should not fall below97%of the initialrateuntil NAD H

hasfallen from10 to 2^iM, even thoughthe ratefrom3to 2\iMshouldbe only 88% of the initial rate.

Fluorometer Indirect Assay(2-50 pmol/min, 0.1-3 nm ol of Product)Reagent:The reagent is thesameasfor Spectrophotometer, except NADH,

10 \LM;ascorbic acid, 2mAf;nicotinamide, 20 mM;andbovine serumalbumin, 0.02%.Procedure

Step1:Place 0.1mLof reagentin 10x75 mmtubes.In a timedsequence,add 5or 10 ILvol of homogenate containing, for example,1ng kidney,0.5 |igliver,and 5 Xgbrain,wetwts.Standardsare 10\iLof 50-300\iMNAD+. Incubate 60 minat20.Step2:Add 10\iLof \M HC1 at thesame rateasthe samples were added;hold 15 minormoreat roomtemperature.Step3:Add1 mL6MNaOHcontaining 10mAfimidazole;heat 20 min at60.Cooland drytubes,and readinthe fluorometer.Cycling Assay(0.2-2 pmollmin, 10-100 pmol of Product)

ReagentThe reagent isthe same asforCycling Assay, exceptNADH,1mM; nicotinamide, 10mM;andbovine serumalbumin, 0.05%.Procedure

Step1:To0.5|iLofreagent under oil are added samples(25-200ng drywtof brainorslow-twitch muscle), 60 min at 20.Step 2: To each sample is added 1 nL of 0.05MHC1;10 min at roomtemperature.


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264 Passonneau and LowryStep 3 : Transfer 1 jiL into a 10 x 75 mm tube containing 100 pL of NADcycling reagent with enzyme levels to provide 500-fold amplificationin1 h at 38.Step4:Complete the assay as described in Chapter 5.

CommentTh e nicotinamide is added in Fluorometer Indirect Assay and Cy clingA ssay to preven t the enzymatic destruction of the product, NA D + . Theascorbic acid is present to prevent the conversion of N AD H to N A D +during incubation and acidification in very small volumes.

M odifications in Cycling Procedurefor Frozen-D ried M uscle Sections(1-5 pmol/min, 100 pmol of Product)

Reagent:Tliereagentis the same asfor Spectrophotometer, except dihydroxy-acetone-P,200\iM;100\xMNADH;and bovineserum albumin, 0.05% .Preincubation reagent: Imidazole buffer, pH 7.0 (30 mAf imidazole base,20 mAf imidazole-HCl);KC1,0.6Af;and bovine serum albumin, 0 .05% .ProcedureStep 1: To 1 pL of preincubation reagent in oil wells are added musclesamples (15 ng dry wt); 30 min at 20C.Step2:Add 4 pL of reagent 1; 60 min at20.Standards are 4\iLof25and50\iMNAD+.Step 3: To each sample is added 5 |xL of 0.1 M HC1; 10 min at roomtemperature.Step 4: Transfer 1 \iLinto a 10 x 75 mm tube containing 100 fiL of NAD

cycling reagent with enzyme levels to provide 1000-fold amplificationin1h at 38.Step 5 : Complete the indicator step as described in Chapter 5 .G l y c o g e n P h o s p h o r y l a s e (E C 2.4.1.1)

(3,5,14,16,29,30,31)(Glucose), (glucose)^ [Re. 7-20]^ r JP PGM

Pi glucosc-1-P -* glucose-6-P 6-P-gluconolactoneG6PDH

NADP+ NADPH + H+


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Collection of Enzyme Assays 265The auxiliary enzym es are P-glucomutase (PGM) and glucose -6-Pdehydrogenase (G6PDH). The glucose-1-P generated may be con

verted to6-P-gluconateas shown, combining all three reactions in asingle step. Alternatively, the auxiliary sequence is carried out in asecond step, which avoids some of the dangers from other tissue enzym es.Phosphorylase exists in two forms; Theb form requires5 AMPforactivity, and the phosphorylated form ais active in the absence of thenucleotide. The kinetics of thea form are complex, and5\AMPcanalter the reaction rate at nonsaturating substrate concentrations (29).Therefore, a very small amount of5fAMPis included in the m ethodsfor phosphorylase a.Sample tissue activities (mol/kg dry wt/h, 20): dog myocardium, 2; humanbrain, 0.50; rabbit soleus muscle, 0.8;and rabbitsoleus muscle, 25.

Tissue HomogenatesTissue homogenates are prepared in 10-20 volumes of imidazolebuffer, pH 7 .0, containing 0 .5 mAf dithiothreitol, 5 mAf ED TA, and 20

mAf KF. The com ponents are chosen to keep theaandbforms in thestate in which they are homogenized. Ifthetissue is quick-frozen, theratio oftheatobforms will be close to that ofthein v ivo conditions.Ifthetissues are removed without precautions to preserve the in vivostate, the state of the enzymes will be a function of the delay beforehomogenization. There is a rapid conversion from the b to a formduring anoxia, follow ed byaslow er transformation backto thebform.The reducing agent is essential for phosphorylase activity; the EDT Aprevents the action of the phosphorylase bkinase and, thus, the conversion of phosphorylase bto theaform. The KF inhibits the actionof the phosphatase that converts the a to the b form. (The spectro-photometric method is not recommended for unfractionated tissue ingeneral because the amount of tissue required in most cases wouldcause problems owing to turbidity and competing tissue enzymes.)

Spectrophotometer(1-5 nm ol/min, 15-75 nm ol of Product)Reagent: Imidazole-acetate buffer, pH 7.0 (35 mAf imidazole base, 15 mAfimidazole acetate); glycogen (glucosyl units), 20 mAf; K 2HP0420 mAf;MgCl2,0.5mAf;EDTA,1.0mAf;NADP+,1mAf;glucose-1,6-bisphosphate,2\iM\ dithiothreitol, 0.5 mAf; bovine serum albumin, 0.025%; muscle P-


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266 Passonneau and Lowryglucomutase, 0.6 U/mL (3 jig/mL);andyeastorLeuconostocglucose-6-phosphate dehydrogenase,1 U/mL. For the measurement of phosphory-lasefl,add2\iM 5 AMP;forphosphosphorylaseaplusb9add 1mAf 5'AMP.

Conduct of the AssayAdd the sample to 1 mL of reagent in a spectrophotometer cu vet.

Read at convenient intervals, 1-5 min. The change in OD should belinear with time after the first minute or two.

Fluorometer Direct Assay(0.02-1 nmol I min y 1-10 nmol of Product)Reagent: The reagent is the same as for Spectrophotometer, except theNADP+ is decreased to 100\xM.

Conduct of the AssayTo1m Lof reagentin1 0 x 7 5mmtubes,add1-10 |iL of homogenateequivalentto25-100p,gof brain, wet wt. Read at convenient intervals,1-5 min, depending on the rate of reaction.

Fluorom eter Indirect Assay(15-75 pmol/min, 1-5 nmol of Product)Reagent 1: The reagent is the same as for Spectrophotometer, except theglucose-6-P dehydrogenase andNADP*are omitted.Reagent 2: Tris-HCl buffer, pH 8 (25 mAf Tris base, 25 mAf Tris HC1);NADP% 100 |iAf; glucose-6-P dehydrogenase 0.2 U/mL; and rabbitmuscle glucose-6-phosphate isomerase, 0.5 U/mL 1 ^ig/mL).

ProcedureStep 1: Homogenate samples (1-5 pL) containing, for example, 1 [igoffast-twitch muscle or 50\igof brain, wet wt, are added to 100 pL ofreagent 1 in 10 x 75 mm tubes at measuredtimeintervals,andincubated for 60min at roomtemperature.Standardsare 5\iLof 0 .1-1 mAfglucose-1-P.Step 2: Add 1 mL of reagent 2 in the same time sequence as Step 1. Thetubesareread when the reaction is complete, 10-20 min.

Cycling Assay(0.2-1 pmollminlsample, 10-50 pmol of Product)Reaction vessels: Oil wells.Reagent: The reagent is the same as in Fluorometer Direct Assay.


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Step1:Freeze-driedsamples(e.g.,25 ngof myocardium,100 ngof brain,dry wt) areintroduced throughtheoil into1 nLof thereagentandincubated 60 minat roomtemperature. Standards are1pL of 10-50\\Mglucose-1-Pin thereagent.Step2:Add 2\ihof0.1MNaOH;20 min at80.Step3:Transfer1\\Linto a 10 x 75 mm tubes containing 100 pLof NADPcycling reagent with enzyme levels to provide about 3000-foldamplificationin 1 h at38.Step4:Complete theindicatorstep as described in Chapter5.

CommentsThe excess of EDTAo ver Mg2+in the reagent is to prevent phosphory-lasebkinase activity,but permit theaction of P-glucomutase, whichrequires onlyalow level of Mg2+.Theprocedures givenabove have been used in themeasurement ofphosphorylase in brain tissue. If muscle phosphorylase activity is to bemeasured in frozen-dried sections, the tissue samples must be

preincubated as described below.M odification of Cycling Assayfor Freeze-Dried M uscle (15 ng Dry W t,2-5 pmol/min, 100-300 pmol of Product)

Preincubationreagent:Tris-HCl buffer,pH8.0 (25mM Trisbase, 25mMTrisHC1);0.6MKC1,0.2%bovine serumalbumin,1mM 5'AMP,10 mMK2HP04,and0.5mMdithiothreitol.Reagent:The same asfor Spectrophotometer.

ProcedureStep1:Thefreeze-driedsamples areintroducedthrough oil into1-\iLdroplets ofpreincubationreagent; 30-60min at20.Step2:Add 4 pLofreagent tosamples. Standardsare the same volumeof25and 50] \Mglucose-1-P.Incubate 60 min at20.Step3:Add 5 MLof0.1MNaOH;20 min at80.Step4:Transfer1 pL into a 10 x 75 mm tubecontaining100\iLof NADPcycling reagentwithenzyme levelstoprovide 500-fold amplificationin1hat 38.Step5:Complete theindicatorstep as described in Chapter5.


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268 Passonneau and LowryG l y c o g e n S y n t h a s e ( E C 2.4.1.11) (9,16,21,31,32)

1. (Glucose)n (glucose)^UDP-glucose UDP

2. UDP UTP

[Re.7-21]lactate

H++ NADH NAD*The auxiliary enzymes are pyruvate kinase (PK) and lactate dehy

drogenase (LDH). PEP is phosphopyruvate. The activity is assessedby measurement ofoneoftheproducts,UDP,by means oftheenzymesequence shown. The synthase exists in an unphosphorylated form,synthase i, which is fully active without the presence of glucose-6-P,and a series of phosphorylated forms that are variably dependent onglucose-6-P for full activity. Therefore, when total synthase activity ismeasured, glucose-6-P is incorporated in the reagent. (The spectro-photom etric method is not recommended for unfractionated tissue ingeneral, because the amount of tissue required in most cases wouldcause problems resulting from turbidity and competing tissue enzymes.)

Tissue PreparationCrudehomogenates are preparedby addingappropriatevolumesof 50mAf Tris buffer,pH7.5,containing5mAf EDTA,20mAfKF,and5 mAfdithiothreitol to the tissue and homogenizing in a glass homogenizer.

The tissue preparations can be storedat-70.The EDTA prevents theactivity of the protein kinase, which phosphorylates synthaseiin thepresence ofATP.The K F prevents the removal ofthephosphate fromthephosphorylated enzymebyphosphatase activity presentin thehomo-genate. The reducing agent dithiothreitolensuresreduction ofthesulfhy-dryl groups oftheenzyme, which are necessary for optimal activity.


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270 Passonneau and LowryCycling Assay for Freeze-Dried Tissue Sections

(0.1-1 pmol/min, 10-100 pmol of Product)Reagents1 and2:The reagents are the same as for Spectrophotometer.ProcedureStep 1: The samples (e.g.,1 \igof liver or m uscle or10 jxgof brain, all drywts) are introduced into 0.2 ^iL of the specific reagent in oil wells.Standards are0.1pL of 25-200\iMUDP. Incubate 2 h at 20 -22.Step2:Add 0.5 ^iL of3mAf H2 0 2in 0.15M NaOH; 30 min at 20-25.Step3:Add1 pL of reagent 2; 30 min at 20-25 .Step4:Add5\iLofO.lAfHC1;20 min at 20-25.Step5:Transfer1 pL intoa10x75 mm tube containing 100 pLof NAD cyclingreagentwith enzyme levels to provide500-fold amplificationin 1hat38.Step 6: Complete the indicator step as described in Chapter 5.

G u a n y l a t e K i n a s e (E C 2.7.4.8) 33,34)1) 5 -GMP 5 -GDP

ATP ADP2) GDP + ADP

2 P-pyruvate

2NADH 2 NAD+The auxiliary enzymes are pyruvate kinase (PK) and lactate dehydrogenase (LD H). Note, 2 mol ofN A D+are generated for each m ol ofG M P phospho rylated, because both nucleotide produc ts of reaction 1,G D P and ADP, are substrates for pyruvate kinase (although the reaction is about fivefold faster with ADP than GDP).Sam ple tissue activities (mol/kg drywt/h,20):mo nkey retina, peak

level 7; rat kidney, 0.24-0.45; and rabbit brain, 2.5.Spectrophotometer(1-3 nmol/min, 25-100 ntnol of Product)

Reagent: Imidazole buffer, pH 7.0 (30 mAf imidazole base, 20 mAf imida-zole-HCl); ATP, 1 mAf; 5'GMP, 500\iM; MgCl2, 2 mAf; and bovineserum albumin, 0.05%.

GTP + ATP[Re.7-22]

2 pyruvate 2 lactateLDH


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CollectionofEnzyme Assays 271Reagent2:Im idazole buffer, pH 7.0 (30 mAf imidazole base, 2 0 mAf imidazole HC1); M gC l2 ,2 mAf; KC1,75 mM; NA DH , 10 0-1 50 \iM\P-pyru-vate, 1 mAf; rabbit muscle pyruvate kinase, 3 U/mL (20 ^ig/mL); andbeef heart lactate dehydrogenase, 0.4 U/mL (2 ^ig/mL).

ProcedureStep1:To 50pLof reagent1in 10 x7 5mm tubes are added5-10 jiL of hom o-genate containing, for example, 2 5 -1 00 \igof brain wet wt. Standardsare 10\iLo f 2 and 5 mAf5 GDP.Incubate at room temperature 6 0 m in.Step 2: Add 25 ^L of 0.1 Af NaOH , and heat 20 min at 60.Step3:To each tube add1 mL of reagent 2, and read when reaction is over,

about 30 min.Fluorom eter Indirect Assay

(0.1-O.25 nmol/min, 2.5-5 nmol of Product)Reagent 1: The reagent is the same as for Spectrophotometer, except ATP,100 nAf; and 5'GMP, 100 \LM.R ea ge nt 2 : The reagent is the same as for Spectrophotometer, except P -pyruvate, 20\xM\and NA DH , 5- 10 [lAf.

ProcedureStep 1: To 50 pL of reagent 1 in 10 x 75 mm tubes are added 5 -1 0 nL o ftissue samples (e.g., 5 \igof brain, wetwt);60 minatroom temperature.Standards are 10 ^L of0.1 and 0.2 mAf GM P.Step 2: Add 25 ^L of0.lAf NaOH; heat 20 min at 60.Step 3: To each tube add 1 mL of reagent 2, except for pyruvate kinase.Read in the fluorometer with NAD H giving full-scale fluorescence.Step 4: Add pyruvate kinase, and read after 30 min.

Cycling Assay(5-30 finol/min, 0.3-2 pmol of Product)

Reagent 1: The reagent is the same as for Spectrophotometer, except: ATP,500\iM;and 5'GMP, 75 \iM.Reagent2:Imidazole buffer,pH6.8 20mAf imidazole base,30mAf imidazole-

HC1); KC1, 75 mAf; MgCl2, 2 mAf; P-pyruvate, 10\xM\ NADH, 1\iM\bovine plasma albumin, 0.025%; pyruvate kinase, 3 U/mL (20 p,g/mL);andbeef heart lactate dehydrogenase, 0.25 U/mL 1|Xg/mL).

ProcedureStep1:Samples (e.g., 5 ng offreeze-driedkidney)are added to 1 pLof reagent1 in oil w ell s. Standards are1 \iLof 0.5and0.1 nAf GDP; 60 m in at 20.Step 2: Add 1\iL of 50 mAf NaOH; 30 min or more at room temperature.


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272 Passonneau and LowryStep3:Add5\iLof reagent2 ;60 min at 25.Step4:Add 2 jiL of 0.2MHC1;10 min at room temperature.Step 5: Transfer 5 pL into a 10 x 75 mm tube containing 100\iLof NADcycling reagentwithenzyme levelstoprovide 10,000-fold amplificationin1h at 38.Step 6: Complete the indicator step as described in Chapter 5 .

H e x o k i n a s e ( E C 2 .7 .1 .1 ) (1,3,16^3,35)Glucose + ATP -* ADP + glucose-6-P 6-P-gluconolactone

X G 6 P D H X ^ [Re. 7-23]NADP+ NADPH + H+

The auxi l iary enzyme is glucose-6-phosphate dehydrogenase(G6 PD H ). Two basic me thods are offered; the first com bines the specific and auxiliary steps as shown above. This is suitable for tissueswith high hexokinase activity and therefore relatively free of disturbing side reactions. Th e second method separates the specific and aux iliary reactions by an enzyme-destroying step, thereby eliminatingm ost of the dan ger from side reactions. This also eliminates the prob lem arising from the fact that tissue 6-P-gluconate dehydrogenase isusually pre sent w ith an activity at least as high as that of g lucose-6 -Pdehydrogenase, and so will oxidize a portion of the 6-P-gluconateyielding an uncertain extra amount of NA D PH . This is less disturbingfor fluorometric assays, because the average 6-P-gluconate level during the assay can be kep t to a small fraction of theKm.In the two-stepprocedure, there is sure to be some conversion of the glucose-6-Pformed to fructose-6-P owing to tissue P-gluco isom erase. The refore,P-gluco isom erase is included in the second reagent to ensure that allthe hexose phosphate is converted to 6-P-gluconate.Sample tissue activities (mol/kg drywt/h,20):mousebrain,2;human brain,

3.4; myocardium, 1; rabbit tibialis anterior muscle, 0.2; rabbit soleusmuscle, 0.60; and liver, 0.02 (The spectrophotometric method is notrecommended for unfractionated tissue in general, because the amountof tissue required in most cases would cause problems resulting fromturbidity and competing tissue enzymes.)


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Collection of Enzyme Assays 273Spectrophotometer Assay

Method 1) 2-10 nmol/min, 50-150 nmol Product)Reagent: Tris-HCl buffer,pH 8.0 (50mM Tris base,50mM Tris-HCl);MgCl2,5mM; glucose,2mM; ATP,5mAf; NADP,0.5mM; bovineserum albumin 0.05%; Triton X-100, 0.5%; and glucose-6-P dehydrogenase(yeast orLeuconostocm esenteroides),0.3 U/mL.

Conduct of the AssaySamples are addedto 1 mLofreagentinacuvet,andreadings made every1 or 2 min. The rate is calculatedfromthe linearpartofthecurve.

Fluorometer Direct Assay Method I)0.05-1 nmol/min, 1.5-10 nmol of Product)

Reagent: The reagent is the same asforSpectrophotometer Assay, exceptNADP*isreduced to 100\iM9and glucose to1 mM.Conduct of the Assay

One-milliliter volumesarepipeted into 10 x 75 mmfluorometertubes. The fluorometer sensitivity is set so that full scale is equivalentto 10|iM NA DPH . Afteraninitial reading,thesampleisaddedandreadingsaremadeat1-5-minintervals. The activity is calculated fromthe linearpartof the curve. Reagent without glucose serves asablank.Suitable amountsoftissueare 25 (ig ofbrain,and 100 (ig ofslow-twitch m uscle and heart muscle,wet wt.

Fluorometric Indirect Assay15-150 pmol/min, 1-10 nmol of Product)

Reagent 1: The same asforSpectrophotometer Assay, except NADP* andG6PDH are omitted, and glucose is reduced to1 mM.Reagent2:Tris-HCl buffer, pH8.0 (25mMTris base,25mMTris-HCl);NADP*, 100\iM\EDTA,1mM; glucose-6-P dehydrogenase (yeast orLeuconostoc mesenteroides), 0.3U/mL;andP-glucoisomerase fromrabbit muscle, 0.2 U/mL 1jig/mL).Procedure

Step1:To100 jiLof reagent1,add 10\\Lof tissue homogenate containing,for example, 5jxgbrain or 50\igofmuscle,wet wt, or 10 nLof1 mMglucose-6-P standard. Incubate at 25 for 60 min.Step2:Add 10 ^L1MHC1;65, 10 min.Step3:Add1 mLofreagent2;read after reaction is complete, 15-30 min.


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274 Passonneau and LowryCycling Assay (1-10 pmol Product)

Reagents: Steps 1 and 2 are the same as for Fluorometric Indirect A ssay,except thatNADP*in reagent 2 is reduced to10\iM.

ProcedureStep 1: The freeze-driedsamples (e.g., 10 ng brain, 50 ng liver, dry wt)ar e introduced through the oil in oil w ells into 1 jiL of rea ge nt 1.Standards are1 jiL of 1-10 \iMglucose-6-P in reagent 1. Incubate 60min at 20.Step2:Add1 ^L of 0.1M HCl; 10 min at 80.Step3:Add5jiL of reagent2 ;20 min at room temperature.Step4:Add 2 ^L of0.25MN a3P04; 20 min at80.If NaOH is used at thisheatingstep todestroytheNADP*,there maybeserious loss of NADPHowing to the presence of glucose.Step5:Transfer 2 pL into a10x75mm tube containing 100JILof NADPcycling reagent with enzyme levels to provide 5000-fold amplificationin1h at 38.Step 6: Complete the assay as described in Chapter 5 .

P - H y d r o x y - a c y l C o e n z y m e A D e h y d r o g e n a s e(E C 1.1.1.35) (3,14,16,22)Acetoacetyl CoA B-hydroxyacetyl CoA

IT + NADH NAD+Th e activity is measured by the rate of disappearance of NA D H indirect assays or the appearance of NAD +in indirect proc edure s.

Sample tissue activities (mol/kg dry wt/h, 20): rat kidney cortex, 12;human vastus muscle, 7.5; dog myocardium, 18; m ouse bra in, 2 .5 ;.human brain, 2.0; rabbit tibialis anterior muscle, 1.6; and rabbit soleusmuscle, 43 .Spectrophotometer

(2-10 nmol/min, 20-150 nmol of Product)Re agen t: Imidazole buffer, pH6.1(15 mAf im idazo le ba se, 135 mAfimidazole HC l); NA DH , 100\iM\acetoacetyl coenzym e A , 200\xM\EDTA, 1 mAf; and bovine plasma albumin, 0.05%.


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To1 mLof reagent inacuvet are added 5 -1 0 pL of tissue h om oge-nate (350|xgbrainormuscle, 50 (ig kidney cortex, wet wts). R eadingsare taken every 2 min. Blanks are tissue added to reagent withoutacetoacetyl coenzym e A. Calculations are made from the linear portion of the curve.

Fluorometric Indirect Assay(2-50 pmol/min, 0.1-3 nmol of Product)

Reagent:Thereagent is the sameasfor Spectrophotometer.Procedure

Step 1: To 100 iLof reagent in 10 x 75 mm tubes are added 5-10 pL oftissue homogenate containing an appropriate amount of tissue (0.5 [igofmuscle,2 ngof brain,0.5|xgofkidney,wetwts).Standards are 10 pLof50-300^MNAD+. Incubate 60 min at 25.Step2:Toeach sampleareadded10\\Lof 0.5MHO;roomtemperature 10min.Step 3: To each sample are added 1 mL of 6Af NaOH containing 10 mMimidazole,and heat 20 min at60.The tubes arecooled, dried,and readinafluorometer.

Cycling Assay(0.3-2 pmol/min, 20-100 pmol of Product)Reagent: The reagent is the same as Spectrophotometer, except: NADH, 50\iM;andacetoacetyl coenzymeA,50\\M.

ProcedureStep 1: Each freeze-dried sample is added to 10 ^iL of the reagent in oilwells.Fortissueother thanmuscle, the samples (10-40ngof brain) areintroduced into the reagent droplet directly through the oil. Standardsare 10 nL of 3-10\iMNAD+.Incubate 60 min at 20.Step2:Toeach sampleadd 2 jiLof 0.3MHC1;10 min at roomtemperature.Step 3: Transfer 2 jiL into a 10 x 75mmtube containing 100 jiL of NADcycling reagent with enzyme levels to provide 500-fold amplificationin1 h at 38.Step4:Complete the indicator step as described in Chapter 5.Special note: Ifthesamples are freeze-dried muscle, the tissue (10-25 ng)is preincubatedinoil wells in1 iLof 50mMimidazole buffer,pH7.0,


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276 Passonneau and Lowry0.6Af KC1;and 0.05% bovine serum albumin, 60 min at 20. This isfollowed by Step 1, above.

H y p o x a n t h i n e ( G u a n i n e )P h o s p h o r i b o s y l T r a n s f e r a s e (E C 2 .4 .2 .8 )1) Guanine + P-ribosylpyrophosphate - > 5'-GMP + PPi2) GMP GDPGK

ATP ADP3) GDP + ADP GTP + ATP

p K ^ [Re. 7-25]2 P-pyruvate 2 pyruvate 2 lactate

LDH

2NADH 2 NAD+Au xiliary enzym es are guanylate kinase (GK ), pyrava te kinase (PK ),and lactate dehydrogenase (LD H). Two m oles of NA D +are formed inthe auxiliary enzyme sequence for each mol of GMP formed in the

specific transferase reaction. NADH disappearance offers the mostdirect assay, but N A D +formation would be better suited for the mostsensitive assays. In direct reading assays, as presented here, PK andLD H are allowed to react with preformed AD P, GD P, and p yruva tebefore adding GK.Sample tissue activity (mol/kg dry wt/h, 20): rabbit brain, 0.1.

Fluorom eter Indirect Assay(0.015-0.15 nm ol/min, 0.5-5 nm ol of Product)Reagent 1: Tris-HCl buffer, pH 7.8 (17 mAf Tris base, 33 mM Tris-HCl);guanine HC1,1mAf;phosphoribosylpylophosphate (PRPP),0.25mAf;KC1,50 mAf; M gCl2,2mm;EDTA,0.1mAf; and dithiothreitol, 0.5 mAf.


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Collection of Enzyme Assays 277Reagent2:Imidazole-HCl buffer, pH 7.0 (30 mM imidazole base, 20mAfimidazole-HCl); KC1,20 mAf; MgCl2,5 mAf; P-pyruvate,0.1mAf; ATP,20\iM;rabbit muscle pyruvate kinase, 0.8 U/mL (5^tg/mL);beef heartlactate dehydrogenase, 0.4 U/mL (2 ^ig/mL); and pork brain guanylatekinase, 0.05 U/mL (added after an initial reading on the fluorometer).

ProcedureStep 1: To 5 0 pL of reagent1in 10 x 75 mm tubes, add 10\xLof sample.For brain, samples should contain 25-250\igof tissue. Standardsare 10 ^L of0.05-0.5mAf5'GMP.Incubate at 25 for 60 min; heat at90 -95 for 2 min.Step 2: To each tube, add 1 mL of reagent 2 containing all the enzymesexcept guanylate kinase. Allow 10 min for PK and LDH to react, andthen read on thefluorometeradjusted so that the high standard readingis full scale.Step 3 : Add guanylate kinase in a vol of 10\ih.The tubes are read againwhen the reaction is complete, about 10 min.

I s o c i t r a t e D e h y d r o g e n a s e ( N A D )(E C 1.1.1.41) (36)

Isocitrate a-ketoglutarate+ C 02* , , ^ [Re. 7-26]NAD+ NADH + H+Th e activityismonitored bytherate of appearance of reduced N AD H.

Sample tissue activities (mol/kgdrywt/h,20):kidney,0.5;heart3;andbrain,0.6 (DavidB.McDougal, Jr., personal communication).Fluorometer

(0.1-0.6 nm ol/min, 1-10 nm ol of Product)Reagent: Phosphate buffer, pH 7.0 (30 mAf K 2H P04, 20 mAf KH 2P04);isocitrate,2.5mAf; citrate,18mAf; MgCl2,18 mAf; ADP,4.5mAf; NAD+,2 mAf; bovine serum albumin,0.05% ;and0.18%Triton X-100.

Conduct of the AssayTo one milliliter of reagent in 10 x 75 mm tubes are added tissuesam ples (e.g., 20 | ig m yocardium, 100 igbrain, wetwt).Readings arem ade every 1-5 m in. Calculations are m ad e from the lin ea r po rtio nof the curve.


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278 Passonneau and LowryComment

NADMinked isocitrate dehydrogenase isam itochondrial enzym e;the Triton X-10 0 is used to solubilize it. C itrate is added to ensure thatthe citrate-isocitrate ratio is near equilibrium. A D P is a positive effecto rofthe enzym eand isaddedto yield maximal activity.

Isocitrate Dehydrogenase (NADP)(EC1.1.1.42) 23,37)

ra-ketoglutarate + C02[Re.7-27]NADPH+ H+

The activityisassessedby therateofappearanceofNAD PH.Sample tissue activities (mol/kgdrywt/h, 20): mouse brain, 0.7; kidney,12;and rat cerebellum,0.6.

Fluorometer Indirect Assay0.1-0.5 nmol/min, 5-20 nmol of Product)

Reagent: Tris-HCl buffer, pH 8.2 (60mM Tris base,40mM Tris-HCl);NADP4 , 1 mM; MnCl2,0.2mM; isocitrate,1mM; and bovine plasmaalbumin, 0.05% .Procedure

Step 1: To 5 0 ^iLofthereagentin5x60 mm tubesareadded appropriatetissue samples (4-10p,gofkidney,wet wt)in avolof2jiL. Standardsare 2 p,Lof1 and5\xMNADPH added to the reagent. Incubate 60 minat 3 8. Chillto0.Step 2: Transfer 20-jiL aliquotsto 1 mLof0.05MK2H P04containing1mM EDTAtostopthereaction. Readon thefluorometer adjustedsothat the high standard gives full-scale fluorescence.

CommentRead ings shouldbem ade w ithin30 min to besure thatno signifi

cant further reaction takes place.Cycling Assay

0.03-0.15 pmol/min, 2-10 pmol Total Product)Reagent: Thisisthe sameasfor Fluorom eter IndirectAssay,except NADP*is 100 MM.


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Step 1: Add freeze-dried samples (e.g., 5-20 ng of brain dry wt) at regularintervals to 2 iLof reagent in oil wells, and incubate 60 min at 20.Step 2: Add 5 ^iL of 0.5M NaOH at same time intervals as for the sampleadditions. Heat the oil well rack 20 min at 80.Step3:Transfer 2 jiL into a10x75mm tube containing 100 jiL of NADPcycling reagent with enzymelevelsto provide about 3000-fold amplification in1h at 38.Step4:Complete the indicator step as described in Ch

A Collection of Enzyme Assays

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