J Biol. Chem._d-fucose Metabolism in a Pseudomonad

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Vol. Z-17, No. 7, Issue of nyril10, pp. 2222-2227, 18 72Printed in U.S .A.

D-hcose Metabolism in a PseudomonadI. OXInATION OF D-JUCOSE TO D-FUCOXO-6-LACTONE BY A D-ALDOHEXOST~: DEHYDKOGESASE

(Received for publica.tion, November 19, 1971)A. STEPH EX L)AHIZIS$ ANI) RICHARD L. ANDERSONFwnz the Department of Biochemistry, Michigan State university, East Lansing, Michigan 48823

SUMMARYA soluble, NAD-dependent dehydrogenase which is specific

for D-aldohexoses, including D-fucose, D-glucose, D-galactose,D-mannose, D-altrose, D-allose, Z-deoxy-D-glucose, and2-deoxy-D-galactose, has been purified 335-fold from apseudomonad capable of using D-fucose as a sole carbonsource. Fort y-f ive other sugars and related compoundstested did not serve as substrates and did not affect the rateof n-glucose oxidation. The pH optimum was 8 to 8.5 inTris-HCl buffer and 9 to 10 in glycine-NaOH buffer. Theenzyme was insensitive to thiols and thiol group reagentsand was not activated by divalent metal ions. Representa-tive apparent K, values were 5.8 XnM for D-fucose, 0.86 mMfor D-glucose and 0.08 mM for NAD+. The ,3 anomer ofD-glucose was preferred over the a anomer. D-Fuconatewas isolated as the apparent product of D-fucose oxidation,indicating that the unstable D-fucono-fi-lactone rather thanD-fucono-y-lactone was the immediate product. This wasconfirmed by ,the demonstration that D-glucono-6-lactone,but not D-fuCOnO-y-kXtOIU3 or D-galactono-y-lactone, couldserve as a substrate in the reverse reaction. Thus, i t isconcluded that 8-D-glucopyranose and /3-D-fucopyranose arethe actual substrates for the enzyme.

n-Fucosc (6.dcos)--n-galactose) is widely distributed in nature,occurring as a component ol numerous heterosides and of theantibiotic chartreusin (1). Hydrolases apparently specif ic forthe @-n-fucosidic linkage have been found in the tissues of vari-ous nmmmals (2-4), in the yiaceral hump of the limpet. (n), andiI1 the digestive juices of snails (6). However, despite the com-mon occurrence of n-fucosyl compounds and of enzymes whichcatalyze the hydrolysis of P-u-fucosides, the pathway of n-fucosemetabolism has not been previously dcscribcd for any organism.Although an early report. (7) indicated that the coli-aerogenesgroup o f bacteria could metabolize n-fucosc, recent investigators

* This investigat,ion was supported by Research Grant AI 080GGfrom the National Institute of Allergy and Infectious 11iscasrs,United St.nt,es Public Ilealth Service: Journal Art,icle 5688 fromthe ?vlichigen Agricultural I;xperiment Station.$ Predoctoral Fellow of the Xational Institutes of Health;present address, Department of Chemist ry, University of Cali-fornia, Los Angeles, California 90024.

find that Eschericlzia coli metabolizrs D-fucose very slowly (8) ornot at all (9).

We have isolated a pseudomonad which ca.n utilize n-fucoseas a sole source of carbon and energy. This pa.per providesevidence that one of the initial reactions in the metabolism ofn-fucose in this bacteriuln is the oxidation of u-fucopyranose ton-fucono-&lactone, which spontaneously hydrolyzes to n-fuco-nate (Fig. I), and reports some of the propertics of the o-aldo-hexose dchydrogenase that catalyzes the oxidation. Subsequentpapers report the osidat,ion of n-fucofurnnose to n-fuconate yian-fucollo-y-lactone (lo), the dehydration of o-fuconatc t,o 2-keto-3.deoxy-n-fuconate (II), and the cleavage of 2-keto-3-deosy-u-fuconate to pyruvate and n-lactaldehyde (12). -1 preliminarypresentation of some of these data has appeared (13).

3UTERISLS BND MEl'HODSOryalzisnz-The bacteriunk used in this investigation was iso-

lated in our laboratory by incubating nonsterile commercialD-fucosc in a mineral medium. Subsequent standard bacterio-logical techniques yielded a pure culture of an organism whichcould grow with n-fucose as the only carbon source. The or-ganism is a small , nonmotile, aerobic, n raln-negative rod, andthus is considered to be a pseudomonad. I-ntil further taso-nomic studies have been carried out, we \vil l refer to it by thetriv ial designation, pseudomonad AISU-1. The pnt,hwap ofr,-arabinose metabolism by this organism has been described (14).Culture Conditions-Tile organism xTas grown aerobically inFernbach flasks containing 1 liter of medium. The flasks wereagitated on a rotary sha.ker at 32. The mctliurn consisted of:1.35% nazHPOa.7HLO; O.lScr/, KH#O*; 0.3% (SH&SO,;0.02% Y!gS04.7Ha0; 0.0005(x YeSOd; and 3.27; sugar (u-fucoscunless indicated otherwise). The sugnr was autoclaved sep-arately and added aseptically to the mineral Inedium. Stepwisexldition of the sugar was necessary because 1 i;! sugar markedlyreduced the growth rate. The first portion of sugar (20 ml of a2.501~ (w/v) solut.ion) wa.s added immediately aft, cr inoculat.ion.Six additional 20.ml portions of 25 y0 (w/v) sugar were added atB-hour intervals. Thick cell suspensions (Am = 18 to 22) wereobtailled; turbidity measuremcnt,s were nlade wit,h a ColemanJunior Spectrophotometer on appropriate dilut,iolls o f the cul-tures in 18.mm diameter test tubes. The cells were harvested4 hours a fter fina l addition of the sugar, which wa:: approximately40 hours after inoculation.To determine growth rates on various sugars, cultures weregrown in B-mm diameter culture tubes containing 7 ml of min-

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Issue o f April 10, 1(3X A, X. Ilahms and R. L. ilnrlerson 2223coo-

H:OH(SpOntaneoUS) H&H

r HO:H2 HiOHJ bH,,B -D- FUCOPY RANOSE D- FUCONO-J- D - FUCONATELACTONEFIG. 1. Rcaotion for the oxidation of u-fucose to D-fuconate bythe D-aldohesosc dchydrogenasc~.

era1 medium supplemented with 0.5$; of the test sugar. Thetubes were agitated at an angle on a reciprocal shaker at 32.Turbidity measur(mcnts were made at suitable time intervalson appropriate dilutions.

Prepnrafion 01 Cell Rx&acts--Cells \vcrc harvested by centrifu-gation, washed once bv resuspension in distilled water, and werefinal ly r~uspcndcd inO.10 M sodium phosphate buffer (pH 7.0)unless othertvise illdi(*at.ed. The cells were broken by treatingthe suspension lot. 13 min in a Rsytheoil IO-KHz sonic oscillatorcooled with circulating ice water. The superllatan t fluid rcsult-ing from lo-mill ccntrifugation of the broken ccl1 suspension at40,000 x g was used as the cell extract.

n-dldohezose IMydrogenase Standard Assay-The reactionmixture (0.15 ml) consisted of 2.5 Fmoles o f u-glucose, 0.3 pmoleof X*441)+, 15 pmoles of Tris-HCl buffer (pH 8.1), and D-aldo-herosr dchydrogcnase. ,I control was used in the early stepsof purification to c>orrect for S1DH osidase; this control wasminus n-glucose and cont,ainrd 0.05 pmole of NADH in placeof XX)+. (Later in the investigation, WC discovered thatNA&DH osidsse acti\-i ty could bc eliminated by preparing thecrude extracts in a solution conta,ining 0.15 rntil Z-t.hiocthanoIalld 0.10 M Bicine buffe r, pH 7.4.) The reaction was monitoredat 340 nm with a Gilford model 2400 absorbance-recording spec-trophot~ometer thrrmostated at 25. The rate was constantwith time and proportional to the nlzyme concentration in theranges used. A mlit o f n-aldohesose dehydroacnasc was de-fined as the amount of enzyme that catalyzed the reduction of1 kmole of ;\J=lD+ per min in the standard assay.11 alyticnl ~I,lethods~~educ~ing sugar was dctcrmined by themethod of Sumner and Ho\vell (15). Mdonic acids mere detcr-minrd after conversion to the corresponding la&ones by boilingin 1 N HCl for 5 min. La&ncs were determined as the hy-drosamic acids by the method o f He&n (16). Pyruvate wasdetermilled with lactic acid dehydrogcnasc and NADH, alld bythe calorimetric method of Friedeman a11d Haugen (17), asmodified by &I;\-re and Greenberg (18). Protein was usuallydetermined by the method of Varburg and Christian (19) ; incrude rxbracts and other preparations high in nucleic acid con-tent, the biurct method (20) was used, with bovine serum al-bumin as the stalldard.

Descending paper c~hl,orllato~r:Lphy was performed on What-man No. 1 f&r paper, using the followjug solvents: Solvent 1,water-saturated 2.butanone; Solvent, 2, l~butanol-~~~vater-nj 5;ethanol (5:4: 1). Lactoncs were visualized by their formationof hydrosamic acids (21) ; aldonic acids lvere detcctcd by t,hesame method after itl sifu lactonization by spraying with 0.2 n-HCI and heating in an oven for 15 min at 100.Reagenls--u-Fucosc (22, 23), u-fucollate (24), L-mannosc (25),L-glucose (26, 27), L-galactose (28), L-fructose (29), and (i-iodo-

TA~LI: IPurification of o-ddohexo.se clehydrogenase

Fraction

Ccl1 extract.Prot.arnine sulfate sriper-nat)ant(NHJZSOI precipitate;.Sephadex G-200DEAE-celluloseaCalcium phosphate gel.

VOlUIlX Totalprotein

1111 i %i

6c50 I 4220780 4060

53 1010133 i 109330 ~ 14.1

66 1.7

Total Specific Ansa:.ctivity activity 4260

755 0.186459 0.434243

iI 2.24

0.630.861.171.251.481.58

a The values given for these fractions have been corrected forthe proportions of the previous steps not subjected to furtherpurificationdicated references. 6.Dcoxy-D-al losc and 6-deoxy-D-glucose(D-quinivose) were the gift s of Dr. T. Reichstein, TJl1iversit.y ofBasel, Base& Switzerla.nd. 3,6-T>ideosy~u~galactose (abequose)\\*as the gif t of Dr. Otto Luderitz, Max Plan& Institute , Frei-burg, Germany. 2.hcetamido-2.deosy-D-allose and 2-aceta-mido-2-dcoxy-u-altrose were the gif t,s of Dr. Xl. n. Perry, Na-tional Research Council o f Canada, Ott,awa. D-Allosc, D-altrose,D-sylulose, and lo-glucose-free 2-deoxy-n-glucose were the gift sof Dr. W. A. Wood, Michigan Ht,:lt ,e I-niversit y. Other chem-icals were from commercial sources. D-&Iannose (31) and D-galactose (32) were twice recrystallized before use as substrates.

RESULTSGeneral Observations

Growth. Rates-The organism grelv equally well on u~fucoar,n-glucose, L-arabinose, and u-galactosc, with a generation timeof about 3 hours at 32.

Initial Investigations with Crude Cell fixtracts-In prrliminaryexperiments with crude extracts , nc were unable to detect anymodification of -D-fucose by means of isomerization or cpimeriza-tion reactions wheu assayed by various chemical and chromato-graphic procedures. Furthermore, w could not detect, thephosphorylation of n-fucose or u-fuconate with hTP, nor thereduction of D-fucose with N,%DH or N;\DPH. However, ex-tracts readily catalyzed a n-fucose-tlependeiit reduction of NhD+and NlYDP+. Subsequent, invest.igations revealed the presenceof two enzymes which catalyzed pyridine nucleotidc-depcndelltoxidations of D-fUc0z-X. The purification and characterizationof one of these dchydrogcnnses is described below.

Purification of D-dldohexose DehydrogenaseExtracts were prepared from r)-glucose-grown cells . The

following operations were performed at O-4. X summary o fthe purification procedure is given in Table I.Protarnine XulJate Treatment-To a cell extract containing 0.2M ammonium sulfate was added an amount of 2?> (w/v) pro-

tamine sulfate solution (pH 7.0) t,o give a final concentration of0.33%. L&fter 30 min, the prccipitatc was removed by ccntrifu-gation and discarded.

Ammonium Sulfate Prcactior~ation~The protein in the super-natant, f rom the protamine sulfate step was fractionated by theaddition of crystalline ammonium sulfate. The protein prc-

6-deorv-u-galact,oxc (30) were prcparrd as described in the in- cip,itat,irrg between 30 and 40y0 sat,uration was collect,ed by ccn-

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2224 D-Fucose Metabolism. I. o-Aklohexose Dehydrogenase Vol. 247, No. 7

PHFIG. 2. Effect of pH and buffer composition on D-aldohexose

dehydrogenase. The standard assay was used except that the pHand buffer (0.10 hf) were varied with the dehydrogenase concentra-tion constant. pH measurements were made on duplicate reac-tion mixtures.trifugat.ion and was dissolved in 0.01 M sodium phosphate, pH7.0.

Gel Filtration-The above fraction was chromatographed on acolumn (6 X 60 cm) of Sephadex G-ZOO equilibrated with 0.01 Msodiu m pho sphate buffer (pH 7.0). Fractious (15 ml) were col-lected during elution with the same buffer, and those with thehighest spec ific activity were combined. Prior to the nextpurification step, the combined fractions were reduced in volumefrom 135 ml t,o 15 ml with a, Diaflo ultrafiltration cell (AmiconCorp., Cambridge , Massa chusetts) conta ining a type UM-10membrane.

DRAE-celluEose Chronzatography-DEAE-cellulose (Sigma, ex-change capacity = 0.9 meq per g) was pretreated as recom-mended by Sober et a.1. (33), and was equilibrated with 0.02 Msodium phosphate buffer, pH 7.0. A portion (2 ml) of theSephades G-200 concentrate fraction was applied to a column(3 x 5 cm) of the DEAE-cellulose, which was then washed with60 ml of the same buffer. The protein was cluted with a stepwisegradient of 60 ml each of 0.10, 0.20, 0.30, 0.40, and 0.80 M NaClin the same buf fer. D-Aldohexose dehydrogenase eluted at the0.20 to 0.30 M NaCl range. Fractions containing most of the ac-tivity were combined.

Cal&m Phosphate Gel Step-The pooled DEAE-cellulose frac-tions were adjusted to pH 6.5 with 0.05 N HCl and dialyzed for24 hours against 0.01 M sodium cacodylate buffer (pH 6.5). Toa portion (5.0 ml) of the dialyzed protein was added 1.0 ml ofcalcium phosphate gel (containing 6% solids) prepared as de-scribed by Wood (34). The gel suspension was centri fuged for1 min in a clinical centrifuge and the centrifugate was successivelyeluted with 1.0 ml each of 0.01,0.02,0.03,0.04, and 0.05 M sodiumphosphate buffer (pH 7.0). About half of the act ivi ty was re-covered in the 0.01 to 0.02 M range. This fraction was 335-foldpurified with a 137 over-all recovery of act ivi ty (Table I). Itwas free from detectable (

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Issue of April 10, 1972 A. X. Dahms uncl R. L. Anderson 222sTABLE III

Egect of mixing substrates on o-aldohexose dehydrogenuse activityThe standard assay was used except that the substrate wasvaried. Each sugar was at a concentration of 33.3 mM.

Substrate Rate of NAD reduction

pmoles/min/mg proteinD-Glucose.. 6.8D-Galactose 5.1o-Mamma.. 4.5D-Fucose.. 3.6D-Mannose + D-fucose 4.0o-Mannosc + D-glucose. 5.7D-Mannose + D-galactose 4.8D-Glucose + D-galactose 5.8D-Glucose + D-fucose. 5.0D-Fucose + D-galactose. 4.3

D-fructose-6-l; pentoses, D-xylose, L-xylose, D-l~xosc, n-ribose,2.deoxy-D-ribose, o-arabinose, L-arabinose, and D-xylulose; tri-os&s, DL-glyceraldehydc; polyols, D-mannitol, D-glucitol, nayo-inositol, L-arabitol, n-arabitol, xylito l, and ribitol; disaccharides,maltose, cellobiose, sucrose, lactose, melezitose, turanose, meli-biose, and t,rehalose; trisaccharide, raffinose; and derivafives ofD-a.ldohexoses, D-glucuronic acid, D-galacturonic acid, D-galactose-B-P, n-glucose-6-P, u-glucosamine, Ai-acetyl-D-glucosamine, (Y-methyl-glucoside, 6-deoxy-D-allose, 6-iodo&deoxy-D-galactose,2.acetamido-6-deoxy-D-allose, and 2-acetamido-6-deoxy-r-al-trose. Also, none of these compounds reduced the rate of oxida-tion of 33.3 rnM D-glucose when added at equimolar concentra-t,iolls, further substantiating that they have little or no aff ini tyfor the enzyme.

Anomer Preference-The rate at which the enzyme catalyzedthe oxidation of an equilibrium solution of cr,/?-D-glucose wascompared to the rate at which it catalyzed the oxidation of afreshly prepared solution of a-D-glucose at 15.6 and pH 7.5.Under these conditions, in which the mutarotation of D-glUCOSewould be the rate-limiting step (35, 36), ar,P-D-glucose wasoxidized at a rate 5 times that of a-~-glucose (Fig. 4), indicatingthat the @ anomer is preferred over the cy anomer. Since theproducts of D-glucose and D-fucose oxidation by this enzyme arethe b-lactones (see below), the actual substrates have to be P-D-glucopyranose and P-D-fucopyranose.

Nucleotide SpeciJicity-From a Lineweaver-Burk plot, the ap-parent K, for NAD+ was determined to be 0.08 DIM. NADP+was completely inef fect ive as a cofactor for the D-aldohexose de-hydrogenase at concentrations up to 20 mM. Also, 4 mM NADP+did not inhibit the reaction when added to the standard assay.Activators rind Inhibitors-The effect s of various thiols, thiolgroup reagents, and salts are shown in Table IV. The dehy-drogenase act ivi ty was not affected by thiols or thiol group re-agents. Also, there was no inhibition by 6.7 mM EDTA oractivation by divalent metal ions at 6.7 mu; on the contrary,NH4+ and several divalent metal ions caused inhibition.

St&My-Sephadex G-200 fractions (in 0.01 M sodium phos-phate buffer, pH 7.0) were stable to freezing at -20 for at least6 months.The half-life of the enzyme at 55 was about 40 set (Fig. 5).The heat inactivation profiles o f the enzyme assayed with eithern-fucose, D-galactose, D-glucose, or n-mannose as the substratewere superimposable, which is consistent with all four activi tiesbeing the result of a single enzyme.

-ii7

I4

-

MINUTES MINUTES AT 5!? CFIG. 4 (Iejt). Anomer spec ific ity of D-aldohexose dehydrogenase.The standard assay (0.15 ml) was used except that the substratewas 0.026 Mumole of D-glucose, the Tris-HCl buffer was pH 7.5, andthe temperature was 15.6. The cuvettcs containing the reaction

mixture minus D-glucose were equilibrated to 15.6, and then afreshly prepared solution of or-o-glucose or an equilibra.ted solutionof a&D-glucose was added.FIG. 5 (right). Thermal inactivation of D-aldohexose dehydro-genase.used. A DEAE-cellulose fraction (specific act ivi ty, 13.8) wasThe enzyme (1.8 mg of protein per ml) was heated in 0.01 Msodium phosphate buffer (pH 7.0) at 55. Samples mere with-drawn at time intervals and assayed for D-aldohexose dehydro-genase act ivi ty witho-glucose, D-fucose, D-ma.nnose, and D-galaC-tose as substrates. The rate of inactivation was identical with allfour substrates.T.&ISLE IV

Effect oj various reagents on o-aldohexose dehyokogenase activityReagent Concentrstion Relative activity

None...................p-ChloromercuribenzoateSodium iodoacetate.2-Thioethanol .GSHEDTA.MgClz :MnC12.NH&l.. ..: : .(NHd)zSOdCoClz. :NiC12cuso4. :. .I II.FeSO*CaC12ZnClz

0.50.51.01.06.76.76.76.76.76.76.76.76.76.76.7

100100100100100100100978686513516161210

IclentiJication of the Reaction ProductAttempts to isolate a lactone resulting from D-fUCOSe oxidation

by this D-aldohexose dehydrogenase, using the same techniquesthat were employed for the isolat,ion of D-fucono-y-lactone result-ing from D-fucose oxidation by the L-arabino-aldose dehydrogen-ase (10) were consistently unsuccessful. This suggested that t,heD-fucose oxidation product of the D-aldohexose dehydrogenase-catalyzed reaction was the &lactone, which is known to be un-stable (37-39). Thus, the apparent product of D-fucose oxida-tion by this enzyme would be expect,ed to be D-fUCOUate.

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o-Fucose Metabolism. I. o-Aldohexose Dehydmgenase Vol. 247, so. 7TABLE V

Revers ibility of the u-aldohexose dehydrogenase-catalyzed reactionThe reaction mixtures (0.15 ml) contained 30 rmoles of sodium

phosphate buffer (pH 6.5), 0.26 rmole of NADH, 10 pmoles of theindicated ketone, and 0.37 unit of D-aldohexose dehydrogenase(specific activit.y, 24.5). The lactone solutions were prepared in0.10 M sodium phosphate buffer (pH 6.5) just prior to each assay.

Substrate Rate of NADH oxidationnmoles/min

~-Gl~~cono-6-lacto~~e.D-Galnctouo-y-lactone.. .D-Fueono-y-lactonc . .n-Glucon o-&lactone + D-galactono-y-lac-

4.6510.005

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Issue of April 10, 1972 A. X. Dahms and R. L. Anderson 2227nucleotidc-linked dehgdrogenases that oxidize n-glucose (36, 41,43) and L-fucoee (39).It seems likely that t.his n-ddohcxose dehydrogenase functionsin the metabolism of bot,h D-fUCOse arid ~-glucose, since it is in-duced to a high level by both of these sugars and has a reasonablyhigh aff ini ty for both of them. However, it presumably does notfunction in the metabolism of u-galactose or n-mannose, eventhough it can catalyze their oxidation and has a fai rly highaff ini ty for them; n-galactose induces the enzyme only slightly,and t,he organism does not grow on n-mannose.

REFERENCES1. STAN&K, J., CERNP, M., KOCOUREK, J., END P;\c~~K, J. (1963)?he monosaccharides pp. 403-404, Academic Press, New York2. LEVVY, G. A. (1960) l\aLure 187, 10273. LEVVY, G. A., AXD McAI,L~N, A. (1963) Biochem. J. 87,361-3674. ESTERLY, J. R., STANDEN, A. C., AND PEARSON, B. (1967) J.

Histochcm. Cytochem. 16, 4705. LEVVY, C. A., AND MCALL~N, A. (1963) Biochem. J. 87, 2OG-209G. ;~~.~RxAY, A., GoT,R., AND JARRIGE, P. (1964) Ezperientia 20,4417. FIELD, J. T., AND POE, C. F. (1940) J. Bio l. Chem. 132,473-4768. ADLER, J. (1969) Science 166, 1588-15979. ROT&U N, B., GANE SAN, A. K., AND GUZMAN, R. (1968) J. UoZ.

Biol. 36, 247-26010. D.~HMs, A. S., AND ANDERSON, R. L. (1972) J. Biol. Chem.247, 2228-223211. D~HMS, A. S., AI\D ANDERSON, R. L. (1972) J. Biol. Chem.247, 2233-223712. l)I\~~,~~, A. S., AXD ANDERSON, R. L. (1972) J. Biol. Chem.247, 2238-224113. D~HIUS, A. S., .41p\'~ NDERSON, R. L. (1969) Ped. Proc. 28, 54014. D~HMS, A. S., AND ANDERSON, 1~. 1,. (1969) Biochem. Biophys.Res. Conzmun. 36, 80915. SUMNER, J. B., AND HOT~ELL, S. F. (1935) J . Biol. Chem. 108,51-5416. HESTRIK, S. (1949) J. Biol. Chem. 180,249-26117. FRIEDEMAXN, T.E., AND H~UGEN, G.E. (1943) J. Biol. Chem.

147, 415-44218. S.IYRE. 5'. W., AND GREENBERG, D. RI. (1956) J. BioZ. Chem.220, 787-79619. WARB~RG, O., AND CHRISTIAN, W. (1941) Biochem.2. 310, 38420. GORXALL, A. G., BARD~Y~IM,, C. J., AND DAVID, M. M. (1949)J. Biol. Chem. 177, 751-766

21.22.23.24.25.26.27.

28.

29.30.31.

32.

ABDEL-AKHER, M., AND SMITH, F. (1951) J. Amer. Chem Sot.73, 5859FREUDENBERG, K., AND HIXON, R. hf. (1923) Chenz. Ber. 66,2119SCHMID, H., AND KARRER, P. (1949) HeZv. Chim. Acta 32, 1371MOORE. S.. AND LINK. K. P. 11940) J. Biol. Chrm. 133.293-311SOWDE~, j. C., AND %ISCHE~, H. 0. L. (1947) J. Amer. Chem.Sot. 69, 1963HUDSON, C. S. (1951) J. Amer. Chem. Sot. 73,4498FRUSH, H. L., BND ISBELL, H. S. (1954) Report of the U. S.

Atomic Energy Commission, NBS-3845, as cited in (1956)Chem. Ab str. 50, 827aFRUSH, H.L., ~NDISBELL, H.S. (1962) inR.L. WEIISTLERANDM. L. WOLPROM (Editors), X&ho& in carbohydrate chemis-tru. VoZ. I, v. 127. Academic Press. New YorkMa%, J. W:,*AND ANDERSON, R. L. 11968) Carbohydrate Res.8, 344RAYMOND, A. L., AND SCIIROEDER, E. F. (1948) J. Amer. Chem.Sot. 70, 2785ISBELL,H.S.,ANDFRUSII, H.L.(1962)inR. L.~HISTLERANDM. L. WOLFROM (Editors). Methods in carbohudrate chemis-try, Vol. I, p. 145,~Acadcmic Press, New York.WOLFROM,M: L., ANDTHOMPSON, A:(1962)inR.L. WHISTLERAND M. L. WOLFROW (Editors). Methods in carbohvdratechemistry, Vol. I, p. 120,Acaden& Press, New York L

33. SOBER, H. A., GUTTER, F. J., WYCKOFF, X., ills PETERSON,E. A. (1965) J. Amer. Chem. Sot. 78, 75634. WOOD, W. A.. (1955) Methods Enzymoi. 2, 21235. NELSON, J. M., AND BEEGLE, F . M. (1919) J. -Amer. Chem. Sot.41, 55936. STRECKER, H. J., AND KORXES, S. (1952) J. Biol. Chem. 196,76937. DREW, K. I I., GOODYE~~R,E. H., BND HA~ORTH, W.H. (1927)

J. Chem. Sot. 123738. ISBELL, H. S., AED FRIXX, H. L. (1933) J. Res. Mat. Bur. Std.11. 64939. SCHACHTER, H., SARNEY, .J., MCGOIRE, E. J., AND ROSEMMAN,6. (1969) J. Biol. Chem. 244, 4785-479240. OKAMOTO, K. (1963) J. Biochem. (Tokyo) 63,34841. LEE, C. K., AND DOBROGOSZ, W. J. (1965) J. Bacleriol. 90.653-66042. CLINE, A. L., AND Hu, A. S. L. (1965) J. Biol. Chem. 240,4488-

4492, 4493449 7, 4498-450243. AVIGAD, G., ALROY, P., aivn EXI~ARD, S. (1968) J. Biol. Chem.243, 1936-194144. SADOFF, H. L. (1966) Methods Enzymol. 9, 10345. WALLENFELS, K., AND KURTZ. G. (1966) Methods hmym ol. 9,112

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