Alkaline phosphatase from Echinococcus multilocularis: purification and characterization

pp. 253-258,1991

' H O S P H A T A S E F R O M E' ~OCULARIS: PURIFICA'[ CHARACTERIZATION

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CIRON,* W. HAMOUD, G. AZZARJ" and e et de Pathologie Exotique, Universit6 C 8 France; and -uTJiJ a . ~ y u t t ~,..A,~.Z,~A VO~ Jt t a x l t ~ , ¢ t t t u tLaboratoire i x . ~ t u n . w t c t t o l t ~ de ~tl Biochimie UM 38 0024), Universit6 Claude Bernard Lyon I, Lyon, Frs

OCCUS D

,8 avenue Rockees (LBTM CNRS, )0 97 07)

(Received 15 April 1991) )urified alkaline Abstract--1. Kinetic and physical parameters of purifiex anita multilocularis metacestodes, livers of infected gerbils and control cc 2. Km value for p-nitrophenyl phosphate was about 0.05 + 0.02 mM 3. ;:max values were 357 + 67 nmol/min/mg proteins for fo metaccst( 6.7 + 0.8 nmol/min/mg proteins for liver enzyme of infected infectec and contr 4. M, and pI were different for the parasite and hepatic enzyme. 5. The parasite enzyme was less sensitive to the elevation elevatior of temper 6. The isatin inhibition was a competitive inhibition type : parasite ype for liver enzyme.

INTRODUCTION

tom Echinococcas nined.

md 6.7+ I.I and pectively. ~aticenzyme. titivetype for host

fi was found to inflictsome damage on E. multilocularis meta¢ metacestodes (Dclabre-Defayolle et al., 1989).

Le external membranes of the parasite constitute a primary mary component of the host-parasite interface. The~e chemical and physical characteristics of these .'mbranes are important in the host-parasite remembranes ionships (Arme and Pappas, 1983). With the objeclationshi rive to increase the biochemical knowledge of Echinococcus multilocularis larvae (Cestoda), we ve studied alkaline phosphatase, orthophosphorichave ( ), a meremoester vhosohohvdrolase monoester phosphohydrolasee (E.C.3.1.3.1). brane-bound enzyme. Duriez-Vauce ucelle et a[. (1983) 'z-Vaucelle have observed an importantLt enzymatic activity of alkaline phosphatase in the Cestoda. ~estoda. This enzyme has been demonstrated in a n u mLber b e r of hhelminth species, )f helmin and plays an important role in nutriment nutrin triment al absorption, particularly in sugars (Waltz :z and Schardein, 1964; ld Guraya, 1978; Fujino Hart et al., 1977; Parshad and et al., 1983). Before studyinlg its possible role in the )chemical absorption mechanism, a biochemica aical study of alkaline phosphatase was carried out. The purpose of the present ~nt investigation was to conduct a preliminary study of the kinetic and physical parameters of alkaline phosl~ osphatase ofE. muiti[ocularis metacestodes, livers of"infected infected :ted host and livers of control animals, in order ,'r to note the possible ;ite enzyme and the endifference between the parasite zyme of the host liver. In addition Ldition the repercussions of an inhibitory molecule onn the three purified enzymes were studied. Isatin, indoline-2,3 dione was chosen because of its known ~m inhibitory effect on alkaline phosphatase activiv ty (Kohli et al., 1962; Kumar, 1979), In a previous in rive study, this drug

Extraction of hepatic tissue from control amd infected animals Livers of infected animals and livers of control animals were washed several times and homogemized in the same buffer that was used for the metacestod cs extraction. The extraction of alkaline phosphatas¢ ~hatase was conducted as described by Tr6panier et al. (1976).

*Author to whom correspondence lee should be addressed. Abbreviations used: pNPP, p-nitrophenyl sodium dodecyl sulfate; Tris, Trisaminomethan.

Affinity chromatography in Concanavalin A The last supematant was apl applied to column Con dlibrated with 100 mM nM NaCI, 1 mM MgC12, ~e column was washed with

Thq

MATERIALS AND METHq HODS Parasites The Savoie strain E. multilocularis larv~ were maintained ~r/s larvae 3-rn in 3-month old Mongolian gerbils (Meri Meriones unguiculatus ). The g¢erbils were infected with 50 mg of metacestode n inocuT1 postlure by I~ intraperitoneal inoculation. Three-months dislocation infection, c infecti,. the animals were killed by cervical and metacestodes were collected (Delab Dvlabre-Dcfayolle et aL, 1989). Extraction of metacestode alkaline phoslJ~hatase All procedures were carried out at +-4°C. Metacestodes obtained were washed 3 times with 0.9% 0.9 e, NaCI and homogenized in 100raM Tris-HC1 buffer, pH 7.6, 100 mM NaC1, I mM MgCl2, 1 mM CaC12, 0.02 mM ZnC12 using a scissor homogenizer (Virtis "S2Y') for 10min, a grinder sonicated for (Polytron) for additional 10 w.in, and then | 5rain (15W). The homogenate was mixed mi with an equal volume of 1-butanol, cooled to -20°C, and shaken slowly for 45 min. The mixture was then centrifuged centrit at 9000 g for 30 rain in a refrigerated Sorvall centrifuge ge (Rotor S 34). The aqueous layer was removed. The supem ~¢matant fraction and the pellet were again mixed and an equ~ ual volume of 1-butanol was added, then shaken for 455 rain min and centrifuged at 9000g for 30rain.

M. E. SARCIRON et al. s were eluted from the g/ml) dissolved in the ighest spec. act. were tration in an Amicon rane. d at +35°C in a final of Tris-HC1, pH 8.5, ped by the addition of hydrolysis measured at udies in the presence of NPP were treated with atin, 2.2 ml of buller and 0.1 ml ol pNPP 4 m l o f 5 0 m M ofisatin at +35°C for 10 min. The reaction as started by the addition of 20 #1 of purified enzyme :eparation (spec. act. = 290 nmol/min/mg proteins). The ilour of isatin did not affect the enzyme activity assay as te addition of NaOH to stop the reaction caused a change colour from orange to yellow which did not interfere with te results when read at 420 nm. The enzymatic activity was dculated by using a molar absorption coefficient of 75 x 104 M/era (Klumpp and Schultz, 1990). Protein concentration was estimated using the procedure Bensadoun and Weinstein (1976), with bovine serum bumin as standard. The effect of pH on the enzyme activity was observed by ring the following buffers of 0.2 M strength: pH 5.2-8.6 ?ris-maleate), pH 7.8-10 (borate) and pH 9-12 (glycineaOH) with all other conditions remaining the same. milady, the reaction mixture was incubated at different t ~ lmperatures lJ (20-60°C). The Michaelis' constant Km was calculated from Lineweaver-Burk plots. Gel filtration The experiments were conducted on a Sephacryl S-300 SU tperfine column (1 x 60cm) equilibrated with 100mM Tris-HC1 ris-HC1 buffer, pH7.6, 100mM NaC1, 1 mM MgC12. hromatography was carried out by upward flow at Chromato The standard proteins and their mol. wts were: 12~.ml/hr. ml al~oferritin horse spleen, 443,000; catalase, 240,000; aldolase, 158,000; albumin, bovine serum, 66,000; ovalbumin, 45,000; chymotrypsinogen A, 25,00¢ 5,000; cytochrome c, 12,400. ----

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Electrophoresis SDS--PAGE under reducing condition ions was performed on :onditions phoresis unit using a Trisa LKB 200 L vertical electrol~ tescribedby ~ed b Laemmli(1970). buffered discontinuous system described ]%) was performed in presThe gel of polyacrylamid (10.8%) •e boiled for 4 min at 100°C ence of 1% SDS. Samples were in 2% SDS and 5% 2-mercaaptoethanol, then gel electrophoresed at 200 V constant voltage for 4 hr. The startdards and their subunit mol. wts ,ts were: myosin from rabbit muscle, 205,000; fl-galactosidase Lase from Escherichia coli, 116,000; phosphorylase B, 97,400; 400; bovine serum albumin, 66,000; ovalbumin, 45,000 and carbonic carbonic anhydrase, 29,000. hie anh~ The gels were stained for alkaline line phosphatase activity by incubating them at 25°C for 1.5-2hr in 0.1 M Tris-HC1 M /Lnaphtyl acid phosphabuffer, pH 8.6, containing 3 mM aC12 and Fast Blue BB salt tase, 1 mM MgC12, 1 mM MnC1. (1 mg/ml). Protein was stainedd with Coomassie Brilliant Blue R 250 (Jeyaseelan et aL, 1987). Isoelectric focusing on polyacrylamide ,lamide gels All experiments were performed led usin using the 2117 Multiphor system LKB with the 103 power ~er supply LKB. The polyickness) were prepared using acrylamide gel plates (2 mm thickness rlsson Davies, Ohman and the method described by Karls Andersson (LKB Application Note 1973). The pH range of Ampholine was wick was soaked in I M sodium hydro~ wick in 1 M phosphoric acid. The sal

proteins were pr Isoelectric focus power (P = 30 voltage = 1400~ trichloacetic acic water and then methanol, 25% phosphatase act The determin; according to the (1976).

ced 3 mm from the cathode. reed for 90 min at constant ge and intensity (limiting e fixed during 30 min in 12% ased 3 x 10 min in distillated )25% Coomassie Blue, 1% [)% acetic acid. The alkaline ded as described above. [-I gradients was carried out escribed by Trrpanier et al.

Reagents

Biochemicals Bit able and were t phosphate (pN phosl phosphate and phosl were purchased MO); Coomas, amide, amid, ampholir Pharmacia, Upp Phan

of the highest purity availwing sources: p-nitrophenyl e BB salt, fl-naphtyl acid mol. wt standard mixture Chemical Co. (St Louis, 250 from Merck; acrylin A-Sepharose from LKB-

LTS The ~ctivity of alkaline phosT[ specific phatase from [es was about 50-fold :eater than tt hepatic tissue of control greal and infected 67.5 + 5.4; 1.3 _ 0.2; 1.5 + _h 0.5 nmol/min/mg proteins, respectively. re Pr Prior to the determination of kinetic ki~ and physical para ~arameters of enzyme of parasite and ar livers of control and parasitized animals, different procedures of enzyme ot (Table 1). The zymc purification were carded out ~hatase of metaceselution profile of alkaline phosph elutiq todes from Concanavalin A S e p~harose was reptode: resented in the Fig. 1. An excess of alkaline phosphatase activity was deposed on the top of the phos column. The purification factor was wa about twice that colul for parasite I enzyme than for livers live~ of control and infected animals. The yield from different steps of extraction of alkaline phosphatase from the parasite of control or was about half that from hepatic tissue ti infected animals. The determination of kinetic pal ~arameters included reaction with the determination of the Km of the 1 p-nitrophenylphosphate as substrate. A representation of a Lineweaver-Burk plot was obtained (see p-nitrophenyl phosphate Figs 2 and 3). The K m of p-nitro] from metacestodes, livers of infected infe and control animals were identical at 0.05 _+_ 0. mM. However + 0.02 the Vm~x value of enzymatic reacti :tion was higher in metacestodes than in hepatic tissue, 3 5 7 _ 67, 6.7 + 1.1 and 6.7 + 0.8 nmol/min/ t/min/mg proteins, respectively. ~timum pH of functionThe determination of the optimm aarasite and for the ing for the purified enzyme of pal livers of control and infected animads found that they were not significantly different: 8.8, 8 . 6 , 8.6, respect~erature of the reaction ively. The variation in temperatur ranged between 20 and 60°C (Figg. 4). The purified enzyme of parasite, and of liver of control and infected animals have the same opl3timal temperature of parasite of reaction, 35°C. However, the enzyme el preserved about 55% of residual activity at phosphatase activity of and infected animals ~rent mol. wt of purified .

aline phosphatase from Echinococcus mult f alkaline phosphatase from E. muhilocularis me he enzyme activity was assayed with p-nitrophen] as described in Materials and Methods

Supernatant fraction Con A-Sepharose Liver of infected animal Crude extract 50% butanol Supernatant fraction Con A-Sepharose

Total protein (mg)

Specific activity (nmol/min/mg)

520 410 144 2

67.5 83.2 155.6 1625

of infected he substrate

Yield

(%)

97 64 10

416 352 30 1.8

1.3 1.5 6.67 55.6

96 36 18

481 496 39 2.3

1.5 1.6 3.4 60.9

!03 21 18

zyme of metacestode, livers of control and infected imals determinated by gel filtration on Sephacryl 300 was approximately 240,000, 306,000 and 6,000, respectively. The subunit mol. wts observed the SDS-PAGE were calculated and found to 80,000 for metacestode enzyme and 151,000 : hepatic enzyme of control and infected anids. However, isoelectric focusing has shown it the metacestode enzyme was focused with two proximity aximity bands between pI values of 4.7 and 4.8, andd two bands at the origin of the sample, which c o rrresponds tes to the enzyme which has not migrated, The te host liver enzyme gave only one band at pI 4.5 (Fig. 5). Thl The Lineweaver-Burk plots of parasite and control en~r ~jmes are represented in Figs 6 and 7 both in the absence senee and presence of different concentrations of isatin. The s~ type of plot 1: was obtained from infected Ilae same liver enzyme. The Km value of metacestode enzyme increased with the augmentation Iation of isatin concentrations but Vm~ remained unch; nchanged. The Km value of host hepatic tissue of controls rols and infected animals

was 0.05 ( mM ,~ isatin concentr~ augm naentation ¢ natiol of inhil nation 18.2 + _~ 2.1 mM/ fected hepatic ~ fected

modified with different Vm~ decreased with the entrations. The determit Ki was 1.6 + 0 . 5 and ~stodes control and inectively. DISCUSSION

AI~ Alkaline phosphatase seems to Ibe an important enzyn rme for the E. multilocularis met ,~tacestode because the enzymatic el activity is 50 times greater than the phos[ )hatasic activity in hepatic tissue tissi of control and infected animals~ Thl different steps involved in the The th purification of the m metacestode and host hepatic en enzyme have led to the cconclusion that the parasite enzyme behaves differently to the host hepatic tissue. Indeed, Trtpa epanier et al.'s (1976) method alpplied to extract alkaline phosphatase from the livel liver of control and infected animals could not be appli died to the extraclion of parasite alkaline phosphatas )hatase. To extract the metacestode enzyme, it was necessary necest to use two

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;. 2. Double-reciprocal plot of the initial reaction velocity ) vs p-nitrophenyl phosphate (pNPP) concentrations m E• multilocularis metacestodes. Velocity was expressed as nmol/min/mg proteins, :cessive treatments with 1-butanol for longer riod of contact than that used for hepatic enzyme. ds fact could signify that the parasite enzyme was ongly linked to the membrane lipids and that was t observed with hepatic enzyme. However, the Dtion of the parasite enzyme fixed by Con -Sepharose was not obtained using 1-O methyl-Dlcopyranoside and methyl ~-D-mannopyranoside described in the literature. This elution was, in fact, v v tained ~AAA~ using Concanavalin A. Already these data indicate Jicate the difference between the parasite enzyme andd that of host hepatic tissue which can be eluted in habitual conditions. The Km values for p-nitrophenyl phosphate were ide;ntical for the three purified enzymes. The enzyme's affinit inity for the substrate was great. The Km value of 12-da, -day old Hymenolepis diminuta, was 10 times higher ~n that of metacestodes (Pappas, 1982). The E. than multiloa dtilocularis metacestodes had a higher affinity for p-nitrophenyl phosphate thann H. diminuta. However, Hipkiss and Branfordwhitee (1985) have demonstrated for the same 21-day old parasite, that the Km value for p-nitrophenyl phost osphate was about the same as that of ccstode. These aese data show that the enzyme affinity for its substrate rate varies in relation to the growth of the parasite.• The K m value of dog

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0

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Fig. 4. 4 Thermal multilocularis mel multik

(ll, trol animals a: incubated at the incub~ alkalir phosphal alkaline Methods. Meth¢ Enzym~ obtai the activity ac activity activit obtained resents resent: the mean

:aline phosphatase from E. livers of infected and con; of purified enzyme were aperature and assayed for described in Materials and alculated as a percentage of perature considered on the nperature. Each point repnples which were incubated fly.

hepat (Van Belle, 1972). This )atic tissue i value is comparable to those obtained for gerbil obt hepat ~atic tissue. The Vm,~values were very different for metactcestodes and livers enzymes. The Tb speed of recognitior of substrate by the alkaline phosphatase was nition 'me than easier using the parasite enzyme tha using the liver. T h i s was an activation phenomeno lomenon. Th, study of the temperature var variation has shown The that the t metacestodes enzyme seems seem less sensitive to incre~~ses in temperature; at 60°C, 55% of parasite enzymatic activity were still conserved• We were unabl to record any enzymatic ac activity for hepatic unable These differences in enzyme at this temperature. The, thermosensitivity may distinguish tl the alkaline phosphatase according to the specific corigin of enzyme (Harris, 1989; Koshida and Wahrel Wahren, 1989; Chuang, 1990; Klumpp and Schultz, 1990). The mol. wts of purified metaces r~etacestode enzyme and of its subunits, determined by g el filtration and note that the paraSDS-PAGE, have enabled us to nc three homogenous site enzyme was composed of th alkaline phosphatase subunits of mol. wt 80,000. The aikr has a trimeric strucof E. multilocularis metacestodes ha, tissue of control and ture. The enzymes of hepatic tissu structure consisting infected animals have a dimeric str of two homogenous subunits. interesting results Isoelectrofocusing has shown il with the presence of two bands for the parasite only one band for enzyme at pI 4.7 and 4.8, and on infected animals. This hepatic tissue of control and infect difference could indicate different ilsoenzymes in the ;uish by isoelectrofoparasite. It is possible to distinguisl cusing the different molecular forms of alkaline phos1989). This technique phatase (Koshida and Wahren, 198~ constitutes an indirect means for the study of the genom itself. The differences in pI observed can be triation. Each isoenexplained inI__I terms of allelique varia ............. . . . . . . . . . J_ ~ _ ^ to a particular protein a different allele for each ine phosphatase showed

:aline phosphata~ from Echinococcus mui

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Fig. 5. Isoelectric focusing of E. muhilocularb metacestode todes alkaline phosphatase (A), liver qof controls (13) and liver of infected gerbils (C) after Concanavalin A-Sepharose Acolumn. The gel was stained for alkaline phosphatase activity as described in Materials and Methods. t wvo o different isoenzymes of host hepatic tissue. The

heterc :terogeneity of alkaline phosphatase enzyme may be dute to the difference in number of sialic acid and SOgars which are linked to the molecule, or may be due te to the difference in the formation of enzyme-lipid complex (Tr~panier et aL, 1976; Koshida and Wahren 'ahren, 1989; Komoda et al., 1981; Higashino et al., 1989). The inhibition study by 83 mM isatin of the three purified enzymes has shown that isatin induced a fall of about 50% of parasite en~~ n e activity and 30% of control and parasitized hepatic atic tissue alkaline phosphatase activity. Obviously, isatin showed a different

inhibbit.ion mechanism when applied applie( to parasite and hepal mtic tissues enzymes--competiti~ ~etitive and uncompetRive inhibition, respectively. Moreover, Morec the Ki of the meta~ metacestode enzyme for isatin was w,' about lO-fold great reater than for the host hepatic tis )atic tissue enzyme. This meall that the parasite enzyme was more sensitive to means the action a of isatin than was the liver li enzyme. This important result underlines an iml~)ortant difference between the metacestode and host hepatic liver enzyme. In the future this work will w be continued studying the influence of other knc)wn inhibitors on alkaline phosphatase activity of pa mrasites and gerbil hepatic tissue, thus enabling the structure str~ of enzymes to be determined. A study of the possible role of alkaline phosphatase in the absorpt )tion of nutriment,

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of the effect of substrate aline phosphatase; 0 (Fq), 2 aM isatin (0).

M. E. SARCIRON et al. rs (Michelson a n d 983; R o u b a t y a n d visaged. ~n some differences nzyme. These data of target molecules

a was supported by

REFERENCES

me C. and Pappas P. W. 0983) Host-parasite interface. in The Biology of the Eueestoda (Edited by Arme C. and ?appas P. W.), Vol. 2, pp. 297-310. Academic Press, ~,ondon. asadoun A. and Weinstein D. (1976) Assay of proteins in he presence of interfering materials. Analyt. Biochem. 70, M1-250. uang N. N. (1990) A heat-stable alkaline phosphatase "tom Penaeus japonicus Bate (Crustaeea: Decapoda): a ~hophatidylinositol-glycan anchored membrane protein. ~omp. Biochem. Physiol. 95B, 165-169. labre-Defayolle I., Sarciron M. E., Audin P., Gabrion C., Duriez T., ParAs J. and Petavy A. F. (1989) Echinococcus nultilocularis metacestodes: biochemical and ultrastruc:ural investigations on the effect of Isatin (2-3 indolineiione) in vivo. J. Antimicrob. Chemother. 23, 237-245. triez-Vaucelle T., Platteeuro-Bouthemy F., Deblock S. md Petav' Petavy A. F. (1983) Rrsultats prrliminaires de l'rtude and mzymatique d'un anti#ne frais de Echinococcus multilocenz ularis. daris. Bull. Soc. Parasit. Fr. I, 101-104. Fujjino T., Threadgold L. T. and Ishii Y. (1983) Phosphatases ases ultrac ultracytochemically observed in juveniles and adults O)f Fasciola hepatica. Jap. J. Parasit. 32, 1-12. Harris .rris H., H. (1989) The human alkaline phosphatases: what we mow and what we don't know. Clin. Chim. Acta 186, know 133-150. .rt R. J., Turner R. and Wilson R. G. (1977) A biochemiHart cal and ultrastructural study of the mode of action of epis nana. Int. J. Parasit. 7, Bunamidine against Hymenole~ 129-134. oshikazu H., Imanishi H., Higashino K., Muratani K., Toshikazu Amuro Y., Yamamoto Y., Furuyama J., Hirano K., Hong, Y. M., Shimokura M.,, Hirano T. and Kishimoto kaline phosphatases. PurifiT. (1989) Gene structure of alkaline cation and some properties ofFthe fast migrating alkaline phosphatase in FL-amnicon ceils (the Kasahara isoenzyme) and its cDNA cloninlg. Clin. Chim. Acta 186, 151-164. ite C. J. (1985) Ca 2+ inhiHipkiss J. B. and Branfordwhite me phosphatase activity in bition of brush border alkaline rasitenkd. 71, 759-763. Hymenolepis diminuta. Z. Parasitenkd.

Ld Kon O. L. (1987) PolyJeyaseelan K., CI is; Genes and Proteins. A acrylamide Gel ;d Techniques in Molecular Laboratory Ma Press. Biology, pp. 10 E. (1990) Alkaline phosKlumpp S. and lia and cell bodies: purifiphatase from cation and char 'iochim. Biophys. Acta 1037, 233-239. Kohli R. P., Sare M. K. P. and Gujral M. L. (1962)Anticow ffindolin-2,3-dione (isatin). Indian J. Physi , 145-149. Sekine T. (1981) Multiple Komoda T., Sak forms of humar dine phosphatase. Chemical d circulating clearances of and enzymatic ~es. Clin. Chim. Acta 117, fastfast- and slow167-187. (1989) Oncodevelopmental Koshida Koshiq K. and ttase. Enzymatic heterogen)ression of al expt n. Acta 186, 255-264. eity of seminor Kumar n of rat testicular phosphaKuma P. (1979) enon. Enzyme 24, 152-157. tase. tase A non-alh age of structural proteins Laemmli Laemr U. K. [aead of bacteriophage T4. during the ass~ duri Nature Nat~ 227, 65 • (1973) Increased alkaline Miche Michelson E. H. td hemolymph of the snail pho: )hosphatase in I with Schistosoma mansoni. Biomphalaria Bim g, Comp. , 763-767. Con Biochen is diminuta: partial charac)as P. W . (19 Pappa ush and solubilized alkaline terization of th( teri2 brush border )hosphohydrolase activities of the isolated iso pho )lasma membrane. Expl Parasit. 94, 80-86. plas Pappas P. W., Narcisi E. M. and Virginia R. (1983) Alterations in brush border membrane ~mbrane proteins and memAlt¢ ~eworm, Hymenolepis brane-bound enzymes of the tapew brm diminuta, during development in the tt definitive host. Molec. Biochem. Parasit. 8, 317-323. Parshad (197[ Phosphatases in Parsh~ V. R. and Guraya S. S. (1978) helminths: effects of pH and various chemicals and anheln lctivities. Vet. Parasit. 4, thelmintics on the enzyme activities thell 111-120. Roubaty C. and Portmann P. (1988) Relation between Roub~ intestinal alkaline phosphatase activiqy and brush border phosphate, o-glucose membrane transport of inorganic ph and o-glucose-6-phosphate. Pfliigers Arch. ges. Physiol. 412, 482-490. Stinson R. A. (1976) Tr6panier J. M., Seargeant L. E. and St molecular properties of Affinity purification and some molec human liver alkaline phosphatase. Biochem. J. 155, 653-660. inhibition of alkaline Van Belle H. (1972) Kinetics and inh Biochim. biophys. Acta phosphatases from canine tissues. Bio~ 289, 158-168. Waltz J. A. and Schardein L. (1964) Histochemical cestodes. J. Parasit. 50, studies of four cyclophyllidean cesto( 271-277.