I ndia n Journ al of Experimental Biol ogy Vol. 4 1, M arch 2003 , pp. 205 -2 10 Studies on kinetic properties of acid phosphatase from nuclei-free rat liver homogenate using different substrates Ji gnes h D Pand ya, Bhaves h D Pal el, Sub ha sh D Katewa & Surendra S Katyare* Department of Biochemistry, Faculty of Science, M. S. Uni ve rsity of Baroda, Vadodara 390 002, India Received 10 Jll ly 2002: revised 2 Decelll ber 2002 Kinctic propcrti cs of rat li ve r ac id phosphata se we re evaluated using th e conventional sy nthet ic substrat es sod ium beta glycerophos phat e and p-n itrophenyl ph osp hat e (PNPP) and ph ysiologi ca ll y occurring phos phate esters of ca rbohy- drates, vitamins and nu cico tid es . The ex tent of hydrol ys is vari ed depending on the substrat es; ph osp hate esters of vitamins and carbohydrat es were in ge neral poor substrat es. Kin etic analysis reveal ed th e presence of two components of th e enzy me for al l th e substrat es. Component I had l ow Kill and low V'll a, ' Oppos ite was true for component II. The Kill va lues were gen - eral ly hi gh for PNPP and adenos ine diphosphate (A DP). Amo ngs t th e nucl eo tid es subs trat es AMP showed high alTin- ity i .e. l ow K ill ' Th e increase in enzy me activity in general at high substrate concentration seems to be due to substrate bind- ing and posit i ve cooperati vity. AMP which showed high es t affin ity lVas inhibitory at high concentration beyond I 111M. The res ults suggest th at ill sifll th e nu cl eo tides ma y be th e preferred subst rates for acid ph os phata se. The pivotal stu dies of de Ou ve and co ll eag ues led to th e characteri zati on o f Iysoso mes as th e membrane- limit ed sub ce llul ar or ga nell es which contain ac id hy- drol ases t - 3 . It ha s now bee n recogni ze d th at th ese or- ga nell es in a ce ll contain about 70 different types of ac id hydrol ases 4 . 5 . In th eir earlier work de Ou ve el al. demonstrated th at mos t of th e ce llular acid phos- ph ata se ac ti v it y (EC 3.1. 3. 2) conce ntr ated in th is sub- ce llul ar or ga nell e!. 3; sod ium beta glycerophosphate wa s used as th e substrate for demonstrating th e ac id phos ph ata se acti vity l.3 .The enzyme al so hydro- lyzes the nonspec ific phosphata se substrate p-nitro- ph enyl phosphate (PNPP). It is now recogni ze d th at ac id phos ph at ases are a famil y of enzy me th at is widespread in nat ure and are fou nd in many an i ma l and plant sys tems 6 . However, de spite consider ab le amou nt of work , th e function of acid phos ph atase is not yet f ull y understood 6 Never- th el ess , acid phosphatases hav e ga incd importan ce as clini ca l diagnos ti ca l tool s in th e detec tion o \" gy na eco- logical cond iti ons, me tast as izing prostate ca ncer, bone co ndi ti ons i ncl udi ng rh eu mari c os teoblastoma, bone cancer or met as ta si s, osteogenes is i mperfecta, I i ve l' di se ases such as Go ucher's di sease, hyperparath yro id- ism and chron ic r ena l rai lure 6 - tt . "' Corrcspondent author: Phonc: +9 1-0265 -2 795594 Fa x: +9 1-0265 -2 787556 E- mail: pand yajigncs@ hotmail.com Th e Iy soso mes function in situ as scavenging or- ga nell es and help in deg radation of mac romol ec ul es of ce llul ar ori gin and from invading microorga nisms. The process invol ves formation of phagolysosomes or endocytic ves icl es in the ce ll int eri o r. Th e latter ves i- cl es th en fu se with primary Iysosomes with given Function to form secondary Iysosomes, which are con- . I d b I . fl' .. t 3 17 I SIC ere to e tl e Stte 0 cata yt lc activ it y '" -. t may hence be anticipated th at under physiologic conditions th e enzyme ac id phosphata se may be inv o l ved spe- cifi ca ll y in th e dephosphorylation of nat urall y/p hysio- logi ca ll y occ urrin g phosphate es ter s. Th erefore stud- i es of th e kinetic properties of th e enzyme employing naturally occurring phosphate esters rather th an with conventionall y used sy nthetic substrates such as or PNPP are des ir ab l e. T he kin etic properti cs of ac id phosphat ases from plant and fungal so urces have bee n desc ri bed 1 3- 16. However, th ese are not relevan t in th e co nt ex t of lysosomal function in th e animal ce ll. Thus in dep th studi es on th e kinetic properti es of lysosoma l ac id ph osphatase C rom animal so ur ce are des irable. With a view to illu strating this point in prelimin ary st udi es th e kinetic properti es of lysoso mal ac id pho s- pha tase from r at li ve r have been examin ed . For th ese stu di es and PNPP ha ve bee n employed as the . I b 11 t4t 7 tX 1' 1 I ' conventtona su str ates .. .. W l tC 1 are sy nt l ett C nonphysiologi ca l substrates, as reference po ints. Ad- ditio nally, studi es using phosphate es ters o\" carbohy- drates, vitamins and th e pu rine nucl eo tid es have al so bee n undert aken.
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Indian Journal of Experimental Biology Vol. 4 1, M arch 2003 , pp. 205-2 10
Studies on kinetic properties of acid phosphatase from nuclei-free rat liver homogenate using different substrates
Jignesh D Pandya, Bhavesh D Palel, Subhash D Katewa & Surendra S Katyare*
Department of B iochemistry , Facult y o f Science, M. S. Uni versity of Baroda, Vadodara 390 002, India
Received 10 Jll ly 2002: revised 2 Decelllber 2002
K incti c propcrt ics o f rat l i ver ac id phosphatase were eva luated using the conventional sy nthet ic substrates sod ium beta
glycerophosphate (~GP) and p-n i tropheny l phosphate (PNPP) and physio logica ll y occurring phosphate esters of ca rbohydrates, vitamins and nucicotides . The ex tent of hydrol ys is varied depending on the substrates; phosphate esters of vitamins and carbohydrates were in general poor substrates. Kineti c analysis revea led the presence of two components of the enzy me for al l the substrates. Component I had low Kill and low V'lla, ' Opposite was true for component II. The Kill va lues were gen
eral ly hi gh for ~GP, PNPP and adenos ine diphosphate (A DP). Amongst the nucleotides substrates AMP showed high alTin i ty i .e. low Kill ' The increase in enzy me activ ity in genera l at high substrate concent rati on seems to be due to substrate binding and posit i ve cooperati vit y. AMP which showed highest affin ity lVas inhibitory at high concentrat ion beyond I 111M. The results suggest that ill sifll the nucleotides may be the preferred subst rates for acid phosphatase.
The pivo tal studies of de Ou ve and co lleagues led to the characteri zati on o f Iysosomes as the membranelimited subce llul ar organelles which contain ac id hydrolases t-3 . It has now been recogni zed that these organell es in a cell con tain about 70 di ffe rent types of acid hydrol ases4
.5
. In their earlier work de Ouve el al. demonstrated that most of the cellular acid phosphatase acti vity (EC 3.1.3.2) concentrated in th is subcellular organelle!.3; sod ium beta glycerophosphate
(~GP) was used as the substrate fo r demonstrating the ac id phosphatase acti vity l.3 .The enzy me also hyd rolyzes the nonspec i f ic phosphatase substrate p-nitropheny l phosphate (PNPP).
It is now recogni zed that ac id phosphatases are a famil y of enzy me th at is w idespread in nature and are fou nd in many an ima l and plant sys tems6
. However, despite considerab le amou nt of work , the function of acid phosphatase is not ye t full y understood6 Nevertheless , acid phosphatases have ga incd importance as clini ca l diagnosti ca l tool s in the detecti on o \" gynaecolog ical conditi ons, metastas izing prostate cancer, bone condi ti ons i ncl udi ng rheu mari c osteob lastoma, bone cancer or metas tasis, osteogenes is i mperfecta , I i ve l' diseases such as Goucher 's di sease, hyperparathyro idism and chron ic rena l rai lure6
The Iysosomes functi on in situ as scavenging organell es and help in degradat ion of macromo lecules of ce llul ar ori gin and from in vad ing microorganisms. The process invol ves formation of phagol ysosomes or endocy tic ves icles in the cell interi or. The latter ves i cles then fu se with primary Iysosomes w ith given Functi on to form secondary Iysosomes, which are con-. I d b I . fl' .. t 3 17 I SIC ere to e tle Stte 0 cata yt lc act i vity '" -. t may
hence be anti cipated th at under physiologic conditi ons the enzyme acid phosphatase may be involved speci f ica lly in the dephosphorylation of naturall y/phys iolog ica ll y occurring phosphate esters. Therefore studies of the kinetic properties o f the enzyme employ ing naturall y occurring phosphate esters rather than w ith conventionall y used synthetic substrates such as ~G P or PNPP are des irab le. T he kineti c propert ics of ac id phosphatases from plant and funga l sources have been descri bed 13- 16. However, th ese are not relevan t in the contex t of lysosomal functi on in the animal ce ll. Thus in depth studi es on the kineti c properti es of lysosoma l ac id phosphatase Crom anima l source are des irable.
With a view to illustrating thi s point in preliminary studies the kineti c properti es of lysosomal ac id phosphatase from rat li ver have been examined . For these
studies ~GP and PNPP have been employed as the . I b 11 t4t 7 tX 1' 1 I ' conventt ona su strates . . .. W l tC 1 are synt l ett C
nonphys io log ica l substrates, as reference po ints. Additionall y, studi es using phosphate es ters o\" carbohydrates, v itamins and th e pu rine nucleotides have al so been undert aken.
206 IN DI AN J EX P BIOL, M A RCil 2003
Materials and Methods [;11 ::..1'111 (: source - A dult male albino rats o f
Charl es- Fos tcr strain weighing 200-250 g were used. The animals were kill ed by decapitati on and the li ve r was quickly excised and tran sferred to a beaker containing chilled (0°_4°C) 0.25 M sucrose. The tissue was minced, washed repeatedl y to remove adhering blood and then homogeni zed in chill ed 10mM NaC! solut ion usi ng a Potter EI vehjem ty pe glass-Teflon homogeni zer to obtain 10% (w/v) homogenate. The homogenate was subjected to centrifugation at 650 g for 10 min. to remove nuclei and ce ll debri s and th e post-nuclear frac ti on was used as the source of the enzyme. It is ant ic ipa ted that the hypotonic 10 mM
aC! so lution would lyse the lysosomal membranes and thus release all the enzyme ac ti vity.
Assay 0/ acid pl/Osplw/{{se acli l'itl' - The assay medium contained in a total volume of 0.4 ml , O.IM sod ium acetate buller p H 5 and the substrate concentrations as indica ted in the indi v idual ex periments. Post-nuclear supern atant (50 pi Cit. I mg protein ) was used as the source of the enzy me. After preincubati on at -:17°C for 2-3 min , the reac ti on was initiated by add ing the subs trate and a/'ter 10 min of incuba ti on the reacti on was termin ated by adding 0.1 ml of 10% (w/v) SDS so luti on Cor all the substrates excep t fo r PNPP. The released inorganic phosphoru s was es ti mated by the meth od of Fiske and Subba Row
l1) When P PP was the substrate the reaction
was terminated by add ing 2 ml o f 0.2 N NaOH and the amount of jJ-n itropheno l reie;lsed was es timated by measuring the absorbance at 420 nm lx.
For substrate kineti cs anal ys is the concentration of the indi vidual substrate was varied over the range of 0.1- 10 mM; for AM P, the rangc was 0. 1-1 111M. Kill and Vlll~X va lues were determined from the LineweaverBurk and Ead ie-Hol'stee plots20 The data were compu ter analyzed using Sigma Plot version 5.0.
The enzy me ac ti vity is ex pressed as nmoles o f Pi or P P released/hr/mg protein .
Protein es timati on was accord ing to the method o f Lowry e l a121
. w ith bov ine senJln albumin used as the standard .
Results and Discussion In the initi al experiment the relati ve el'fi cacy of the
ac id phosphatase to hydro lyze the vari ous phosphate es ters, both syntheti c and natural was tes ted. In these experiments the substrate concentration was kept fixed at 5 mM. The results are gi ven in Tabl e I . As can be seen, o f the two synthetic substrates, the eni'.yme hydro lY7.cd PNPP w ith a two-fold hi gher ellicacy.
Thiamine pyrophosphate (TPP) was a poor subs trale
whereas the results .v ith ribofla v in 5'- lllonophosphate (FM ) were variabl e in th at detectab le ac ti vity \V,tS
noted onl y in some experiments. Thi s is not surpri sing because TPP and FM are known to be catabol ized
I· 1' 1 1 1-1"0 1 by cytoso IC enzymes w ll C 1 act near neu tra p --'-' . A mongst the phosphate esters of' the carbohydrates
tested, fructose 1,6-b isphosphate (FBP) was hydrolyzed w ith highes t effi cacy foll owed by glucose 1-phosphate (G I P) and inos itol hexaphosphori c ac id ( IHP). Results w ith glucose-6-phosphate (G6P) were of variab le nature. In thi s con tex t it is interes ting to note th at G6P is hydro lyzed by microsomal enzyme which has a pH optimum of 6.5 24
; incubati on at pH 5 for 10 min is known to inacti va te the microsomal glu cose-6-phosphatase5
.
Of th e three nucleotides tested highes t and lowest ac ti v ities were seen respecti ve ly with ATP and AMP; activi ty w ith ADP was intermediate.
In view of the result s in Tabl e I the substrate kinct
ics parameters w ith PNPP, I3GP, FBP ane! the three adenine nucleotides were in vesti gated. The substrate concentrati on range emp loyed for these stud ies was O. I- IOmM.
The typi ca l substrate sa turati on curves for these substrates are shown in Fig. I . As is ev ident cha racteri sti c substrate sa turati on plots were seen for all the substrates up to a concentrati on of 10 111M except for AMP where maximum acti vi ty was noted at I 111M concentration. Concentrat ions of A MP beyond I mM were inhibitory (eg. see FigA). Therefore separate experiments were carried out where maximum A MP
Tab!..: I - Activity or ral l i ver lysosomal acitl phosphatasl.! (nnlOIcs/hrlmg protein ) w ith sy lllhclic and natural subsl rates .
I Valucs arc Illcan ± SE or thc nu mber or indcpcndent observation, g ivcn in parcill ilcses l
G roup
Carboil yd ralcs
N ucll.!o tidcs
V ilamin deri val i vcs
Substratc
PI PI' [:lGP
FBI' G I l'
IHP
G6P
ATP
ADP
AMP
T PP
17M
Enzy me ac t ivilY
2237.0 ± 177.49 (:-;)
11 73.9 ± 11 2. 10 Ul)
763.8 ± 270.3 (5) 526. 3 ±-WA (9)
246.5 ±-I9.2 (7)
va riablc rcsults (X)
232 1.4 ± 162.:5 ( 10)
180-l.0±210.3 (e)
682.7± 69.7 (5)
8-1. 3 ± 18.8 (Xl va riablc result s (X)
...
PAN DY A elll/: KI ET IC PROPERT IES OF AC ID PHOSPHATASE 207
concentration was I mM and data po ints were increased by includ ing add iti onal nine substrate concentrat ions over the J mM range.
The Kill and V illa, values were determ ined from the corresponding Lineweaver-Burk and Eadie-Hofstee plots20 For sake of brevity onl y the typi cal EadieHofstee plots are shown in Fi g. 2.
The inhi bitory effect of higher concentration by A MP was analyzed in terms of Murray plot (Fig. 4) which is described separately .
As can be seen from the typica l Ead ie- Hofstee plots, for all the substrates tested the ac id phosphatase act ivity reso lved in two components. Component I had low K ill and V illa, and Component II had high Kill and Villa" T hc K ill and VII"" va lues calculated from the Li neweaver-B urk and Ead ie-Ho fstee plots were in close agreement and these va lues were averaged. T he mean of averaged values of K ill and Villa, arc given in T able 2.
3 5 00 14 00 A B
1200 2800
10 00
2 10 0 8 00
14 00 6 00
4 00 700 -
2 0 0
0 0 0 6 9 12 0 3 6 9 12
3 500 35 00
C
28 00 2 8 00
21 00 210 0
>-14 00 14 0 0
u 0
7 00 70 0
'" E
"" 0 0 N 0 4 6 8 10 c: 12 0 3 6 9 12
W 15 0 0 2 0 00
E F
15 00
1000
1000
5 00 5 00
0 ~---'---'1----'----4
0 ·0 0 ·2 OA 0 -6 0 ·8 1·0 1· 2 o 6 9 12
[s], m M
Fi g. I -- Typica l s li bs t r~Il C s~l tu l '; ll i o n cu rves 1'0 1' r<lt live l' Iysoso lll ~1I acid phosph ata sc using d il 'i'crcnt substra tcs. The enl.Y lllc <lctiv ity (v) is pi oiled on ordi nate ag;linst thc corrcs p0<i d ing subst ratc concentra ti on on absc issa. The cxpcrilllcntal dc ta il s are as givc lI in the te,xl. A. I' IPP, B. ~GP, C. AT 1', D. ADP, E. AMP and F. rBP .
As can be noted (T ab le 2) , the va lues of Villa, was about 3.5 times higher w ith P PP th an th at obtaincd
w ith ~GP . FBP was the poorest substrate w ith lowest
value of V llla'J ' The adenine nucleoti des were good
substrates and the ex tent of hydro lysis of the adenine nucleotides increased with phosphory lation.
The va lue of K II,! was relati vely high and about
comparable for PN PP and ~GP as substrates. K IllJ for
FBP was also in the simi lar range. By contrast, the K II 1J va lues for the three adenine nucleotide substrates
were much lower ind icating high af f inity, Amongst the th ree nucleoti des, the lowest K lll J was noted for
A MP whi le the highes t was noted for A DP; the va lue o f K Ill J fo r ATP was in the intermediate range.
The V II""2 was about one and hal f ti mes higher w ith
PN PP as substrate compared w ith ~G P . For the th ree nucleoti des the va lue or V llla '2 decreased w ith the ex
tent of phosphory lati on. Paradoxicall y, however, th e hi ghes t V II""2 was seen for FBP. It may, however, be
30 00 15 0 0 A B
20 00 1000
1000 500
0 0
0 1000 2000 3000 4000 5000 0 4 00 800 1200
3500 3500 >-
0
"" 2 8 0 0 2800
>-
U 2 100 2100
0 1400 14 00
'" E >- 70 0 70 0 N c: W 0 0
0 17 50 3 500 5 2 50 7 000 0 2000 40 00 6000 8000
1500 2500
E F 20 00
1500
10 00 5 00
5 0 0
o -j-----.-----.---"'--j o -j--- .-----.-=="'-----j o 10000 2000 0 3000 0 o 4 00 800 1200
v / [ s J Fig. 2 -- Typical Eadic- Horstcc pl ots 1'0 1' l'aJ li vcr lysoso mal acid phos phat ase us ing d illc rent substratcs. The cnl.y ll1 l: ac ti vity ( v ) is pl ollcd on ordi natl: aga inst thl: COITl:sponriing valucs or v/ lSI 011
absc issa. T hc cx perimcnt a l dc tail s arc as g ivcn in thc Jcx t and in Fig. I. A. I' PI' , B. fIGI' , C. AT P, D. ADP, E. AM P and F. f-'BP.
208 INDIAN J EX P SIO l , MA RC H 2003
borne in mind that the K ill} fo r FBP was also so very
high (8.5 mM; ego see T ab le 2); ph ys io log ica ll y one could never expect to reach such high concentration of FBP ( in the range or so to 60 mM i. e. 6 to 8 times K ill}) which will sa turate component II to g ive the
high Villa,,} value. Bes ides, FBP is metabo li zed cy toso
li ca ll y }5 . Hence, the observed high rate of hydrolys is or FBP by the lysosomal enzyme is unlikely to occur under phys iolog ic conditions. The Kill} va lues were
comparable for PNPP, pGP, ATP an d A DP but were the highes t for FBP as po inted out above, once again emph as izing the fac t th at FBP was a poor substrate. T he Kill} va lues were lowest for A MP.
Since the ac id phosphatase acti vity w i th all the substrates tes ted reso l ved in two components di fferi ng in their Kill and Villa" va lues (T ab le 2, Fig. 2) it was o f interest to find out whether the affinity of the enzy me 1'01' the substrates was concentrati on dependant. T o eva luate th is poss ibility Hi ll p lot analys is}O was carri ed out. T ypi ca l Hill pl ots for the various substrates are shown in Fig. 3. As can be seen the Hill pl ots for all the subs trates were biphas ic. Froill the Hi ll pl ots the Hill coe fri cients n I and n2 respecti ve l y for th e low and high substrate concentration ranges were ca lculated. The concentration o f subs trate at which the transiti on in substrate bind ing pattern occurred was also computed . These data are g iven in T able 3 from which i t Illay be noted that for al l the substrates studied the va lue of nl was close to I indica ting th at in the lower substrate concentra ti on ran ge cooperati ve interact ion was not invo lved. However, at highcr subs trate concentrat ions, except in the case of AD P, the va lue of n2 increascd ind icat ing pos iti ve coopera ti vity. The transi tion po int was in the range of I mM fo r all the substrates except fo r A MP w hich was in the ran ge of
0. 1 mM, a va lue lower by a magnitude. T hi s is consistent w ith the substrate saturati on k inet ics describcd earlier (Fig. 2, T able 2). However, for A MP the substrate concentrati on efrects were of dua l naturc; concentrati ons up to I mM acti vated the en zyme whereas higher concentrations i .e. above I mM inhi bi ted the enzyme (eg. see Fig. 4 ).
Fig. 3-T ypica l Hill pl o ts 1'01' rat li ve r ac id phu;, ph:llase usi ng d irfere nt substrates . The value 0 1' log v/V -v o n u rd in:lle is pl Olled aga in st 10g[S[ o n absc issa 1'01' each su bst rate . The ex pe rilllental
dcta il s a l'e as g ivc n in the text. A. PN PP. S. rJG P. c. ATP. D. ADP, E. AMP and F. FBI'.
Tab le 2 - Kine ti c prope rt ies or ac id phosphatase w ith sy nl he lic and nat ural substrates
[Va lues a re Illea n ± SE or the number or independe nt o bserva tions g ive n in pare ntheses [
KIllI and K"'2 represe nt the Mic hae lis Mc nte ll constal1ls and Villa, I and VIlI:"2 represent the max imu m
ve locity 1'01' the respec ti ve K",s .
Kill = mM, V""" = nillo les/hrlmg prote in
,
PANDYA el (II: KINETI C PROPERTI ES OF ACID PHOSPHATAS E 209
/000 0·00 8 8 > A
,., 750 0 ·006
>
0 ..: 0 -00 4 0 5 00
Q)
E 250 0·002 ,., .. N c:
W 0 0·000
0 2 4 6 8 10 12 0 12
[5), m M [S], mM
Fig. 4 - Typi cal substrOl tc sa tmati on cu rvc (A) and typ ica l Murray plot (13) 1'01' AM P. Thc cnzymc acti vity v is plOllCd on ordinatc aga inst the corrcspond ing substratc concclllrati ons on absc issa 1'01' plot A. In pl ot 13 the va luc or Ilv is plottcd on ordlnatc aga instthc corresponding va lue or lSI on absc issa.
Inhibiti on of enzyme by higher concentration of AMP was analyzed in terms of Murray plot 20 The
typica l Murray plot is shown in Fig. 4, from which the Ki for AMP was found to be in the range of 10 mM. The <1 vera o ed va lue of Ki from 9 experiments was b
10.52 ± 0.46 mM. Phys iologically , however, such high AMP concentration are unlikely to be present. Hence the significance of the inhibiti on of the enzy me by higher AMP concentrations remains obscure.
Nevertheless, the results of the present studies have clearl y shown that the nucleotides are the preferred substrate f or the lysosomal acid phosphatase with AMP showing the highes t affinity i.e. low Kill va lues. It may hence be suggested that in s itll the enzyme may preferentiall y act towards hydrolysis of mononucleo tide such as AMP. A lthough ex periment s using other purine and pyrimidine nucleo tides were not carri ed out. It is possible that even w ith these substrates, the mononucleotides cou ld be the preferred substrates.
It was interesting to note th at the acid phosphatase acti viti es w ith all the substrate tes ted reso lved in t'vvo components. Thi s observa ti on warrants some di scussion. The increase in the enzy me act i vity with increasing substrate concentrati on would suggest positive cooperati ve interact ion20 The data from Hill pl ot analys is (Fig. 3 and Tabl e 3) al so suggest that in general in high concentration r;.lI1ge th e number of substrate molecules bound increased. Nevertheless, as is apparent from Fig. 2, the substrate saturation curves were not sigmoidal imply ing th ~tt allos teric regulation was not in vo lved. Thi s was also substantiatcd by the Lineweaver-Burk plot (data not shown) and Ead ieHofstee plot s (Fig. 2); as is well recogni zed the allosteri c data can not be anal yzed by Lineweaver-Burk and Ead ie-Hofstee pl ots20 Therefo re, the alternate explanation seems to be that the enzy me has an inbuilt abil ity to respond to va ry ing concentrations o f
Tabl c 3 - Hill plot analysis for ac id phosphatasc in rat li ve r w ith di ITe rcnt substratcs
I Va lucs are I1lcan ± SE or th c nUl1lbcr or indcpcndcnt obscrva tions givcn in parcn;hcscs l
A MP(5) 0.537 ± 0. 107 1.91 7 ± 0.536 O. I 15 ± 0.021
FBP(5) 0.739 ± 0.046 1.505 ± 0.084 1.558 ± 0.213
substrate. Thu s the enzyme can hydro lyze the substrate with lesser efficacy if the substrate concentrati on is low and can ac t more effi cientl y if the substrate leve l is higher. Thi s in-built ability will ensure complete breakdown of phosphate esters from phagocytosed macromolecules of ce llul ar ori gin or from in vading microorgani sms, irrespect i ve of the resulting concentration of phosphate esters. Multicomponen t behav ior is not unique to acid phosphatase but has also been noted previously fo r mitochondrial FoFI ATPase as well as microsomal Na+, K+ ATPase by us and other researchers26
.2x
. A lso, the in-built abil ity of the enzyme to act on mono-, di-, and trinucleotide substrates i .e. AMP, ADP and ATP becomes additional hand le for dephosphory lation.
The present preli minary studi es were carried out using the nuclei-free homogenate as the source or the enzyme. Nevertheless, th ese studi es have clea rl y shown that ill situ the nucleotides may be preferred substrates of acid phosphatase. The results or the present studi es al so suggest scope for further experi ments using Iysosomes or Iysosomes-rich fraction as the source of enzyme w hich can shed further li ght on the in situ function of ac id phosphatase.
Refen~nccs clc Du vc C & Watti .:tux R, Functions or Iysoso lllcs, 111/1/11 Re i' Pit v.liul, 28 ( 1966) 435.
2 dc' Duvc C, The m lc or lysosu lllcs in cc llul ar pathology, Trial/gil', 9 ( 1970) 200.
3 BJinton D F, Thc discovery or iysoso lllcs, J Cell liiol , 91 ( 198 1) 66.
4 Dcan R T & 13~:rell A J, Lysosollles, Essays li iocitelll, 12 ( 1976) I.
5 Bal'cll A J & Hca th M F, Lysosoma l cnzy lllcs, in LysosOIl/es: A lobor{/{ory hal/dbook , cd itcd by J T Dinglc (North -Holl and Publi shcrs, Amstcrdam) 1977, 19.
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