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

-tt .

"'Corrcspondent author: Phonc: +9 1-0265-2795594 Fax : +9 1-0265-2787556 E-mail: pandyajigncs@ hotmail. com

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 ).

> I

>

0> o

1·0 -.----------,-~ I · 0 -,--------r--. A B

0 ·5 0 ·5

0·0

0 ·0

- 0'5

-0 ·5 - 1' 0

- I · 5 f----,-----,---.-,--,----j

-1 '5 - 1' 0 -0·5 0 ·00·5 1·0 1· 5 -1 ' 5-1 ' 0 - 0 '50·00'5 1·0 1· 5

1·0 1·5 -.------- --r-, C D

1·0

0 · 5 0 · 5

0 ·0 0·0

-0·5

- 0 ·5 - I· 0 +--,-,---.-,--,----1 - 1·5 -1 ' 0 - 0, 5 0-0 0·5 1·0 1·5 - 1,5 -1'0-0' 50.0 0 '5 1·0 1.5

1·0 0 ·5 ,-------,-----,

E F

0 ·5 0 ·0

0 ·0 -0 ,5

- 0' 5 · ·1 · 0

- I · 0 - I· 5 ¥ -.--,-,--,---r----4 - 2, 5 -2·0-1 ' 5 - 1'0 - 0· 5 0 ·0 0· 5 - 1,5 -1 '0 - 0 ·5 0·0 0 ·5 1·0 1·5

log [5J

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 [

Substrate COlllponen t I COIllPonent II

K ill ] V m:J'(1 K JlI2 V I1U '(2

PNPP(9) 0.331 ± 0.034 IS77.3± 163.1 1. 10 ± 0.097 2965.9 ± 18S.6 ~GP(8) 0.24H 0.030 466.8 ± 65.4 1.93±0.240 1924.5 ± 220.7 ATP (8) 0.088 ± 0.0 I 0 1293.6 ± 126.4 1. 26 ± 0.200 2933.0 ± 138. 1 ADP (S) 0. 156 ± 0.022 12450± 16 1.2 0.SO ± 0.1 17 2247. 0 ±241.0 AMP(S) 0.048 ± 0.007 83S.0 ± 63.4 0.37±0.070 1474.5 ± 152.0 FB I' (7) 0.361 ± 0.056 43 3.7 ± 49.9 S.4S ± 0.630 43 15.0 ± 133.9

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

Substratc Hill Cocrri cicn t Transiti on

nl 11 2 concentrat ion, l1l /\Il

PNPP(9) 0.80 I ± 0.040 1.484 ± 0. 163 1.007 ± 0.179

pGP( I O) 0.557 ± 0.05 I 1.459 ± 0. 103 1.053 ± 0.082

ATP( I O) 0.585 ± 0.05 1 1.1 63 ± 0. 100 1.206 ± 0.2 16

A DP(7) 0.553 ± 0.042 0.96 1 ± 0.053 0660 ± 0.093

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.

6 Bull H, Murray P G, Thomas D, Fraser AM & clson P N, Acid phosphatascs, J Ciil/ Poi/wi: M ol Pm/llil, 55 (2002) 65 .

7 Macicjcwsk i R, Kopi eni ak M , Madcj 13, Sck A & Dabrowski A, Aciel phosphatasc ~lc ti v it y in di lTcrcnt OI"gans as a marker

210 INDI AN J EXP BIOl, MARCH 2003

of acutc pancrcatiti s, A 1/11 Vlli v Mariae Cllrie Skiodolllska (Mer/I, 56 (200 1) 356.

8 Wozniak A, Drcwa T, Rozwodowska M, Drewa G, Lam­brccht W & Wisni ewska I. Ac ti vit y of somc lysosomal cn­zy mcs in scrum and in tumors of pat icnts with sq uamous ce ll lung carcinoma, Neoplasllla, 49 (2002) 10.

9 http://www.fpnotebook.com/HEM63.htm 10 Lad P M, Lcarn D B, Coopcr J F & Reisingc r D M, Dcscrip­

tion of prostatc acid phosphatasc isoc nzy mcs in normal and canccrous statcs, Ciill Chilli Acta, 14 1 ( 1984) 5 1.

II Nakasto Y R, Janck il a A J, Hal lcen J M, Vaa nanen H K, Walton S P & Yam L T, Clini ca l signifi cancc of immunoas­says for typc-5 tartaratc-rcsistancc ac id phosphatasc, Ciill Chelll, 45 ( 1999) 2 150.

12 Szcgo C M & Pc itras R J, Lysosomal fu nctions in ce llul ar activation: Propagati on of thc acti ons of hormones and othcr clTcc tors, 1111 Rev CylOl, 88 ( 1984) I.

13 Olczak M, Watorck W & Morawiecka B, Purifica ti on and characteri za ti on of acid phospha tase fro m yc ll ow lupin (Lu­pinus lutcus) sccds, Biochilll lliophvs Acta, 134 1 ( 1997) 14.

14 Pcnhcitcr A R, DulT S M & Sar;Jth G, Soyabc:t n root nodulc acid phosphatasc, Plalll Physiol, 114 ( 1997) 597.

15 Pcn heitcr A R, Kluc;JS R V & Sarath G, Purificat ion and charactcri zat ion of soyabean root nod ul e phosphatase cx­prcsscd in Pichia pastori s, PrOleill E rpr Pllrij: 14 (1998) 125.

16 Wyss M. Bruggcr R, Kroncnbcrger A, Rcmy R, Fembcl R, Ocstcrhelt G, Lchmann M & Van Loon A P, Biochmica l charac tcri za ti on of fu ngal phytases (myo- inositol hexakis­phosphate phosphohydrolascs):cata lytic propcrt ics , Appl EII­

"iroll Microbiol, 65 ( 1999) 367. 17 Panara F, Angiolill o A, Di Rosa I, Fagotti A, Contcnti S, Si­

moncclli F, Lorvik S & Pasco li ni R, Puri fica ti on and so mc properti cs of Mg (2+) ac ti va tcd ac id phosphatasc from rat tcs ti s, 1111 j /Jiochelll , 26 ( 1994) 885 .

18 Torriani A, Innucncc of inorganic phosphatc in thc fo rmation of phosphorus by Escherichia coli , /Jiochim Biophys Acta. 38 ( 1960) 460.

19 Fiskc C H & Subba Row Y, Colorimetri c detcrmination of phosphoru s, j Bioi Chelll , 66 ( 1925) , 375.

20 Di xon M, Wcbb E C, Thorne C J R & Tipt on K F, Enzy mc kincti cs, EIIZYlll es, (Longman, London) 1979, 47.

2 1 Lowry 0 H, Roscbrough N J, Farr A L & Randa ll R J, Pro­tcin measurcment with the Fol in phenol reagcnt, J Bioi Chelll , 193 (1951 ) 265.

22 Granjciro J M, Fcrrcira C V, Juca M B, Taga E M & Aoya ma H, Bov inc kidncy low molecular we ight acid phosphatasc: FMN-depcndcnt kincti cs, Biochelll Mol Bioi 1111 , 4 1 ( 1997) 120 1.

23 Sipovs kii V G ,Ultracytochemica l study of act ivit y of various phosphatases during carl y postmortcm pcriod in cxperimcn­tal an imals, Vopr Med Khilll , 42 (1996) 23 1.

24 Cra in R C & Zil vcrsmith D B, Lipid dcpc nde ncc of glucose-6-phosphate pyrohyd rolasc: A study with purifi cd phosphol­ipid transfcr protcins and phosphati dy linositol-spccific phos­pholi pasc C, Biochelllislry, 20 ( 1981) 5320.

25 Li Y P, Curley G, Lopez M, Chavcz M, Glcw R, Aragon A. Kumar H & Baca 0 G, Protein tyros inc phosphatasc ac ti vity of Coxiella bllmelii that inhib its human neut rophil s, Acta Vi ­rol , 40 (1996) 263 .

26 Parmar D V, Ahmed G, Khandkar M A & Katya rc S S, Mi­tochondri al ATPasc: a target for pa raccla mol-induccd hepa­totox icity. Ellr j Phan llacol Toxicol Pham wcol Secl ioll, 293 ( 1995) 225.

27 Ccrdan E, Campo M L, Lopez- Morata ll a & Santiago E, Thrcc cata lyti c sitcs in mitochondrial ATPase, FEBS LeI!, 158 ( 1983) 15 1.

28 Kau shal R, Davc K R & Kat ya re S S, Paraceta mol hepatotox­icity and microsomal funct ion, EII\'iroll Toxicol Plw l'l lIocol, 7 ( 1999) 67 .