Characterization of Leishmania donovani acid phosphatases.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc

Vol. 260, No. 2, Issue of January 25, p : &X-@,1985 nnted m (I. S. A.

Characterization of Leishmania donovaniAcid Phosphatases* (Received for publication, August 8, 1984)

Alan T. RemaleyS, Siddhartha DasS, Phil I. Campbell$, Gregory M. LaRoccaS, Michael T.Popen, and Robert H. GlewS1) From the $Department of Biochemistry, The University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, the SDepartment of Biochemistry, The University of Benin, Benin City, Nigeria, West Africa, and the llDepartment of Chemistry, Georgetown University, Washington, D. C. 20057

A crude membrane fraction from promastigotes of Leishmania donovani grown in a liquid culture medium containing 20% fetal calf serum was prepared by freeze-thawing, centrifugation (200,000X g, 30 min), and extraction with 2% (w/v) sodium cholate. After removal of the bile salt by chromatography on a Sephadex G-75 column, the solubilized membrane protein fraction, rich in acid phosphatase activity, was chromatographed on columns containing concanavalin ASepharose, QAE-Sephadex, and Sephadex 6-150 and G-100. Threedistinct acid phosphatases wereresolved: the major phosphatase activity (70% of the total) was L-(+)-tartrate-resistant (designated ACPPI) and corresponds to the acid phosphatase localized to the outer surface of the parasite’splasma membrane; the other twophosphatases (ACP-P2 and ACP-Ps) account for the remaining 30%of the particulate acid phosphatase activity, andboth of these enzymes are L(+)-tartrate-sensitive. Using a combination of sucrose density gradient centrifugation, gel filtration chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was determinedthat ACP-P1 is a 128,000-dalton protein composed of two subunits of 65,000-68,000 daltons. ACP-PI has an isoelectric point of 4.1, a pH optimum of 5.5, hydrolyzes fructose 1,6-diphosphate, but no other sugar phosphates and dephosphorylates phosphotyrosine, yeast mannan, and the phosphorylated form of rat liver pyruvatekinase. ACP-P2 (PI, 5.4) and ACP-Ps (PI,7.1) with molecular masses of 132,000 and 108,000 daltons, respectively, are both tartrate-sensitive andare distinguished from each other on the basis of their sensitivity toinhibition by polyanionic molybdenumcomplexes. These two phosphatases also have their pH optima in the pH 5.06.0 range, but have a considerably broader substrate specificity than ACP-P1.

Leishmania donovani, the etiologic agent of Kala-azar or visceral leishmaniasis, is responsible for significant morbidity and mortality throughout the world (1).The flagellated protozoan has a digenetic life cycle; in the alimentary tract of its insect vector, the sandfly, it exists extracellularly as themotile promastigote form (2), whereas in the phagolysosomal system of mammalian macrophages, it exists intracellularly as the nonmotile amastigote form (3).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed.

Knowledge about interactions between components of the external surface of the parasite and the cell surface of the host is of obvious importance in understanding the complex life cycle of this organism. In 1981, Gottlieb and Dwyer (4) reported the presence of intense acid phosphatase activity distributed over the entire external surface of L. donovani promastigotes. Acid phosphatase activity was also localized to intracellular vesicles. The existence of unusually high concentrations of acid phosphatase activity in L. donovani was later confirmed by Glew et al. ( 5 ) . In the same study, it was also shown that promastigotes contain relatively low levels of other acid hydrolases. After extraction from the plasma membrane and partial purification, the principal acid phosphatase was found to have a pH optimum of 5.5, to be tartrateresistant, and capable of catalyzing the dephosphorylation of fructose 1,6-diphosphate, p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and a variety of other phosphomonoesters. Other members of the Trypanosomatidm family also possess membrane-bound acid phosphatases (6-8). The physiologic role of these acid phosphatases has yet to be established. The present paper reports on the purification and characterization of three acid phosphatases (ACP-PI, ACP-P2, and ACP-Ps) derived from membranes of L. donovani promastigotes. Special emphasis has been placed on ACP-P1, which accounts for more than 70% of the total cellular acid phosphatase activity. EXPERIMENTALPROCEDURES

Organism and Growth Conditions-A cloned strain of L. donovani (Sudan 1) promastigotes were cultivated and harvested as described by Gottlieb and Dwyer (4), except for the addition of streptomycin (50 pg/ml) and penicillin (50 units/ml) to thegrowth medium. Determination of Acid Phosphatase Activity-Acid phosphatase activity was determined routinely using a fluorometric assay, with 4methylumbelliferyl phosphate serving as thesubstrate (5).The standard assay was carried out for 15 min at 37 “C in a 0.1-ml reaction mixture containing 0.2 M sodium acetate buffer (pH 5.5) and 7 mM MUP.’ Enzyme activity toward other low-molecular-weightphosphomonoester substrates was estimated by measuring the release of inorganic phosphate by the method of Lanzetta et al. (9).One unit of enzyme activity was defined as the amount of enzyme required to convert 1nmol of substrate to product/h. Phosphorylation and Dephosphorylation of Purified Pyruvate Kime-Phosphorylation of purified rat liver pyruvate kinase using [y32P]ATPand protein kinase was carried out as described by Blair et al. (10). The 32P-labeledprotein contained 1.6 molof 32P/mol of protein (assuming the molecular weight of pyruvate kinase to be 250,000).To investigate the ability of the purified ACP-PI preparation to dephosphorylate 32P-labeledpyruvate kinase, we incubated 0, 80, 160, and 220 units of ACP-PI for 10 h at 37 “C with 30 pgof The abbreviations used are: MUP, 4-methylumbelliferyl phosphate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid; SDS, sodium dodecyl sulfate.

Characterization of L. dorwvani Acid Phosphatase pyruvate kinase in 0.1 ml of medium containing 0.2 M sodium acetate buffer, pH 5.5. Heat-inactivated phosphatase (5 min 100 "C) was also used as a control for each point. The extentof dephosphorylation of 32P-labeledpyruvate kinase was determined as described by Blair et al. (10). Protein Determinution-Protein concentration was estimated by the method of Bradford (11) using bovine serum albumin as the standard. Isolation and Purification of Acid Phosphatases-All procedures, unless otherwise noted, were carried out at 4 "C in eitherbuffer A (10 mM sodium citrate, pH 6.5, 0.02% (w/v) sodium azide) or buffer B (10 mM sodium phosphate, pH 6.0, 0.02% (w/v) sodium azide). A washed pellet containing 3.1 X 10" L. donovani promastigotes was suspended in 80 ml of 25 mM Hepes buffer, pH 7.2, containing 0.9% (w/v) NaC1, and subjected to three freeze-thaw cycles to disrupt cellular membranes. The crude homogenate was diluted to 100 ml with buffer A and centrifuged at 200,000 X g for 30 min. The pellet, containing approximately 75% of the totalacid phosphatase activity, was resuspended in 50 ml of buffer A supplemented with 2.0% (w/v) sodium cholate. The suspended pellet was then extracted by homogenization in a Potter-Elvehjem homogenizer, followed by stirring for 15 min with the aid of a magnetic stirrer. The crude sodium cholate extract was centrifuged at 200,000 X g for 30 min. The resulting supernatant was set aside, and thepellet was re-extracted twice with sodium cholate as described above. The three sodium cholate supernatant fractions from the bile salt extraction steps were combined and applied to a Sephadex G-75 column (5 X 105 cm) and eluted with 2 I of buffer A. All of the acid phosphatase activity was recovered in the void volume fractions, while the bile salt eluted at the retention volume. Fractions from the Sephadex G-75 column that contained acid phosphatase activity were pooled and loaded onto a QAE-Sephadex A-50 column (Fig. 1). Approximately 10% of the acid phosphatase activity appeared in the column breakthrough fractions (data not shown) and was designated as ACP-P3. The column was then developed with a 4-1, 0-0.6 M linear sodium chloride gradient prepared in buffer A , two peaks of acid phosphatase activity were resolved, and these were designated as ACP-PI(Pool 11) and ACP-P2 (PoolI). Each of the three acid phosphatase pools were chromatographed separately on aconcanavalin A-Sepharose column. The concanavalin A-Sepharose column (2.5 x 8 cm, 10 mg of concanavalin A protein/

TI

60 80 IO0 120 I40 Fraction Number FIG. 1. QAE-Sephadex chromatography of sodium cholatesolubilized L. donovani acid phosphatases. The fractions containing acid phosphatase activity from the void volume of the Sephadex G-75 column were pooled and applied to a column (8 x 18 cm) containing QAE-Sephadex A-50 equilibrated in buffer B. The column was washed with 10-bed volumes of buffer B followed by elution with a 5 1 of linear NaCl gradient (0-0.6 M NaCl in buffer B). Fractions (20 ml) werecollected and analyzed for acid phosphatase activity (O), protein (O), and conductivity (A). The acid phosphatase activity that appeared in the breakthrough was designated ACP-Ps, while pools I and I1 were designated ACP-PZ andACP-P,, respectively. 0

20

40

881

ml gel) was equilibrated in buffer B supplemented with 0.1 M NaCl, 1 mM CaC12,and 1 mMMgC12. The column was eluted with 50 ml of 0.2 M a-methylmannoside. The ACP-P1 preparationfrom the concanavalin A-Sepharose step was further purified by sequential gel filtration chromatography on Sephadex G-150 and Sephadex G-100. MolecularWeight Determinations-Molecular weights and frictional ratios ( f/fo) of enzymes were determined from s % , ~values and Stokes radii (12). Sedimentation coefficients were determined by sucrose density centrifugation as described by Martin and Ames (13), using bovine lactate dehydrogenase as the standard. The enzyme preparations and sucrose gradients were prepared in buffer A, and the gradients were centrifuged at 37,000 rpm for 19 h in a Beckman SW-50.1 rotor. The Stokes radii were determined by Sephadex G-150 chromatography using a column calibrated with protein standards. Isoelectric Focusing-Each acid phosphatase preparation was subjected to isoelectric focusing at 4 "C according to the procedure of Vesterbergand Stevenson (14) using an LKB apparatus(Model 8100). Electrofocusing was carried out for 19 h at 3 watts in a110-ml sucrose density gradient (0-28% (w/v)) containing 0.63% (w/v) ampholytes, pH 3.0-10.0. Polyacrylamide Gel Electrophoresis-Proteins were subjected to electrophoresis in sodium dodecyl sulfate-containing gels according to the method of Laemmli (15), with a 3% polyacrylamide stacking gel and alinear 10-20% polyacrylamide gradient serving as the separating gel. Gels were stained with Coomassie Brilliant Blue G250. Electrophoresis under nondenaturing conditions were performed with a 3% polyacrylamide stacking gel and a 9% separating gel at pH 6.8 and 8.8, respectively. To each sample, 10 pgof bovine serum albumin was added, and the gels were stained for acid phosphatase activity using naphthyl phosphate as the substrate, as described by Lam et al. (16). RESULTS

Fractionation of Multiple Forms of Acid Phosphatase and Purification of the Major Particulate Form-In addition to the excreted extracellular acidphosphatase described by Gottlieb and Dwyer (171, L. donouani promastigotes produce at least three other acid phosphatases. The first evidence that there mightbe more than a single speciesof acid phosphatase associated with the particulate fraction of L. donouani came from an analysis of t h e amounts of total activity and tartratesensitive activity inthe membrane fraction at various points during the growth curve (Fig. 2). It was known from previous studies (5, 17) that the predominant acid phosphatase associated with L. donouani membranes was not inhibited by tartaric acid. In the present study, it was found that the inhibition of acid phosphatase activitybyL-(+)tartrate ranged from 30% in the early log phase t o 15% during the stationary phase. It is noteworthy that tartrate-resistant activity reached a maximum between days 6 and 7, whereas tartrate-sensitive phosphatase activity peaked on day 3. These results prompted us t o monitor the purification for the presence of tartrate-sensitive formsof acid phosphatase. As indicated in the fractionation scheme summarized in Table I, we were able to solubilize and separate three acid phosphatases from the crude membrane fraction.After three freeze-thawcycles and vigoroushomogenization,approximately 75% of the total acid phosphatase activity remained bound t o the particulate fraction. The membrane-bound acid phosphatase activity resisted solubilization when extracted with either 1.0 M NaCl or 10 mM EDTA, suggesting that t h e not activityresidesinintegralmembraneproteins(data shown). After solubilization with sodium cholate, the crude membrane-derived acidphosphatases were resolved into three components by QAE-Sephadex chromatography(Fig. 1). Approximately 10% of the activity broke through the column and was designated ACP-P3. Two peaks of acid phosphatase activity wereeluted from the column with the sodium chloride gradient, and these were designated ACP-PI and ACP-P2.

Characterization of L. donovani Acid

882

Pool 11, corresponding to ACP-P1, represents the major acid phosphatase of L. donovani that islocalized on the surface of the parasite (4, 5, 17). All three acid phosphatases derived from the particulate fraction bound to a concanavalin A-Sepharose column and were eluted with a-methylmannoside, indicating that all three enzymes are probably glycoproteins. ACP-Pl was purified further by chromatography on a series of Sephadex gel filtration columns. The protein concentration in thefractions from the gel filtration columns was belowthe limits of detection, thereby precluding assessment of the coincidence of protein and activity profiles. On a SDS-polyacrylamide gel, the final preparation of ACPPI ran asa pair of sharp andequally intense Coomassie Bluepositive bands with M, = 65,000 and 68,000 (Fig. 3). The sum of the molecular weights of these two proteins (133,000) is close to thenative molecular weight of the ACP-PI(128,000), which was estimated from the results of gel filtration chromatography and sucrose gradient centrifugation.

loBl

Phosphatase

It remains to be seen, however, if both of these bands represent components of the native acid phosphatase or if one of them is a contaminant. The specific activity of the most pure preparation of ACP-P1 estimated, in the presence of phosphatidylserine, was 5.2 x lo6 units/mg protein; this value is similar to that of other acid phosphatases that have been purified to homogeneity (18).We could not assess the purity of the other two acid phosphatase preparationsbecause of the small amount of protein in each sample. Although not indicated in Table I, ACP-P2 and ACP-P3 were each purified further using a concanavalin A column. Even though the protein concentrationsof the phosphatase preparations eluted from the lectin column were below the limits of detection, we nevertheless estimated that the specific activity of ACP-Pp and ACP-Ps exceeded 1.0 x lo6units/mg protein. In addition to the particulate acid phosphatases, approximately 25% of the total acid phosphatase activity appears in the soluble fraction. The soluble acid phosphatase activity was fractionated into three peaks when subjected to DEAESephadex chromatography (data not shown). Preliminary results indicate that these forms are distinct from the membrane-derived acid phosphatases. Isoelectric Points and Electrophoretic Mobilities-The three purified, membrane-derived acid phosphatases were subjected to isoelectric focusing (Fig. 4) and electrophoresis in a polyacrylamide slab gel under nondenaturing conditions (Fig. 5). ACP-P, had the lowest isoelectric point (PI, 4.1 k 0.25) and

=

c

1

3

5

7 9 1 1 Culture Age (days)

1

3

= ”””

- v

FIG. 2. Acid phosphatase activity during the growth of L. donovani promastigotes. A cultureflask containing 200 ml of medium 199 and 20% fetal calf serum was inoculated with 3.2 X lo6 cells. Aliquots were removed at the indicated times, and cell density was determined byhemocytometry.Cells were harvestedand washed, and the crude particulate fractions were prepared as described under “Experimental Procedures.” The particulate fractions were assayed for acid phosphatase activity in the absence and presence of 12 mM L-(+)sodium tartrate from which tartrate-resistant (0)and tartratesensitive (A) acid phosphatase activity was determined.

J

L

- 200 - 116 - 91 - 66 - 45 - 31 - 24 - 14

Anode

FIG. 3. SDS-polyacrylamide gel electrophoresis of ACP-PI.

ACP-PI after the Sephadex G-100 step (lam1) and from the Sephadex G-150 step ( l a n e 2) were analyzed by SDS-polyacrylamide gel electrophoresis and stained for protein as described under “Experimental Procedures.”

TABLEI Purification of acid phosphatases from L. donovani Purification step

Total activity units X 10’‘

Total protein

w

Specific activity units X 10a/mg

70.6 845 8.36 Crude homogenate 17.9 211 8.48 High-speed supernatant 242 15.8 38.3 Cholate extraction 132 26.8 35.4 Sephadex G-75 QAE Sephadex 16.5 33.7 49.9 (a) ACP-PI 9.12 12.3 74.1 (b) ACP-Pz 3.13 3.96 79.0 (c) ACP-P3 3.15 412 13.0 6. Concanavalin A-Sepharose (ACP-PI) 7. Sephadex 6.82 0.26 2623 Gel filtration stem (ACP-PI) a One unit = 1 nmol of methylumbelliferyl phosphate hydrolyzed per h at 37 “C. * Calculated by considering the activity in step 5 (a) as 100%.

1. 2. 3. 4. 5.

Yield Purification

-fold

1 1.70

%

100 92.4

3.16 75.2 26.1 166

78.8’ 41.3‘

Characterization of L. donouani Acid Phosphatase

883

ACP-P1and ACP-P, are much less active at pH 4.0 than at the higher pH. In addition, all threeof the acid phosphatases are stablefor up to1h at 37 “C in the pHrange 4-8 (data not shown); however, ACP-P1,ACP-P2, and ACP-Ps were all rapidly inactivated when exposed to pH3.5. Inhibitors-The three membrane-derived acid phosphatase preparations were compared with respect to their sensitivities to traditional phosphatase inhibitors (Table11).ACP-P1 was clearly distinguished from ACP-P2and ACP-P3by its resistance to inhibition by L-(+)sodium tartrate. ACP-Pa could be distinguished from ACP-P2 by the greater sensitivity of the former to ammonium molybdate and the two polyanionic molybdenum complexes (Table 111);ACP-P3for example, was eight times more sensitive to complex B than was ACP-P,. All three particulate acid phosphatases were markedly inhibited by fluoride ions. The presence of CaC1, (1 mM) and MgC12 (1 mM) had no

Fractton Number

FIG. 4. Isoelectric focusing of the three particulate acid phosphatases. Isoelectric focusing was performed as described under “Experimental Procedures.” A, ACP-P, (1960 units); B, ACP-Pz (1800 units); C, ACP-P3 (12,000 units). Two-ml fractions were collected and analyzed for acid phosphatase activity (0)and hydrogen ion concentration (0). Cathode

Origin

pH

pH

pH

FIG. 6. The effect of hydrogen ion concentration on the activity of the particulate acid phosphatases of L. donovani. The activity at various pH was determined under standard assay conditions using the following buffers: 0.2 M sodium acetate (O),0.2 M sodium cacodylate (0),and 0.2 M Tris-HC1 (A). A, ACP-P,; B, ACP-Pz; C, ACP-P3.

0 7 , Lane 1

Lane 2

TABLE I1 Summary of the effect of various compounds on the activity of ACP-P,, ACP-Pz,and ACP-Pa Addition

Final concentration

Acid phosphatase activity ACP-PI

ACP-Pz

I

1 I L

Anode

FIG. 5. Nondenaturing polyacrylamide gel electrophoresis of L. donovani acid phosphatases. Lanes 1,2,and 3 contain ACPPI, ACP-Pz, and ACP-Pa, respectively. The gel wasstained for activity as described under “Experimental Procedures.”

the greatest anodal mobility at pH 8.8, whereas ACP-P3 (which did not bind to QAE-Sephadex at pH 6.0) had the highest isoelectric point (PI,7.1 k 0.05)and thelowest anodal mobility. ACP-P2 had an intermediate isoelectric point (PI, 5.4 f 0.14) and migrated between ACP-P, and ACP-P3on the polyacrylamide slab gel. Treatment of ACP-PI, ACP-P2,or ACP-P3with either Clustridiurnperfringens neuraminidase or Escherichia coli alkaline phosphatase did not alter the electrophoretic mobility of any of the L. domuani acid phosphatases. Effect of Hydrogen Zon Concentration on Acid Phosphatase Activity-All three membrane-derived phosphatases were true acid phosphatasesin that theyexhibit maximum activity below pH 6.0 (Fig. 6). ACP-P, is distinguished by the fact that it is nearly equally active at pH 4.0 and 5.5, whereas

ACP-Ps

76 control”

mM

None (100) (100) (100) Sodium fluoride 5.0 8.9 f 2.7 6.3 f 0.98 2.1 f 0.66 Cupric sulfate 5.0 57 f 2.1 7.5 f 0.52 9.8 f 2.5 Sodium dithionite 25.0 38 f 10 19.5 f 1.1 8.9 f 0.95 Ferrous sulfate 5.0 53 f 4.1 41 f 13 47 f 2.1 EGTA 10.0 118 f 0.35 125 f 28 120 f 12 KzHPOI 10.0 86 f 4.9 51 f 9.9 39 f 0.71 L-(+)-Tartrate 0.50 94 f 1.4 15 f 1.98 20 f 3.3 Each result is the mean of four determinations f 1 S.D.

TABLE I11 Summary of the effect of polyanionic molybdenum complexes on acid phosphatases of L. domuani Each inhibitor was tested over a range of six concentrations. A plot of log (% inhibition) versus log (inhibitor concentration) yielded a straight line from which the concentration required to inhibit activity by 50% was determined. The values are reported with 95% confidence intervals. B = [C(NHZ)~]Z[(CBH~)ZASMO,O~~H] .HzO; E = 01 - ( N H ~ ) B [ P Z M ~ ~ ~ ~ B Z ] . ~ H Z ~ . Im of molybdenum compounds inhibitors Enzyme

ACP-PI ACP-P2 ACP-PI

B

E

Ammonium molvbdate

3.88 f 0.34 1.93 f 0.21 15.5 f 2.9

0.856 f 0.012 0.254 f 0.37 1.49 f 0.40

16.6 & 2.1 9.33 -+ 1.4 64.6 & 14

Characterization of L. dorwvani Acid Phosphatase

884

effect on the activity of the three membrane-bound acid phosphatases. Energy of Activation-In a study of the effect of temperature on reaction rate that was performed using MUP as the substrate, the energy of activation for ACP-P1, ACP-P2, and ACP-P3was determined from Arrhenius plots to be 7.46,11.2, and 9.16 kcal/deg/mol, respectively. By statistical analysis, the values for the energy of activation were found to be significantly different at thep < 0.05 level, providing further evidence for the uniqueness of ACP-PI, ACP-Pz, and ACPPB. The Effect of Various Lipids on Acid Phosphatase ActiuityEnzymes associated with membranes often exhibit a lipid requirement (19, 20) and, since our purification procedure could have removed membrane lipids, it was of interest to determine if the inclusion of phospholipids or gangliosides in the assay medium would affect phosphatase activity. Specifically, the effects of phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and the ganglioside GMl on the activity of ACP-P1, ACP-P2, and ACP-P3 were compared. In all cases, except for phosphatidylethanolamine, inclusion of the various lipids in the standardassay slightly increased the K,,, for MUP and stimulated phosphatase activity from 1.6to 3.3-fold (Table IV,Fig. 7). The various lipids activated each acid phosphatase to a different degree. For instance, phosphatidylcholine activated ACP-P3 the most, followed in order by ACP-Pl and ACP-Pz,while the monoganglioside GM1 activated ACP-P, the most, followed in order by ACP-PI and ACP-P,. Phosphatidylethanolamine inhibited ACP-Pz activity, while it activated ACP-PI andACP-P,. Size-related Parameters-The molecularweights of the three membrane-derived acid phosphatases were estimated from sedimentation coefficients, obtained by sucrose density centrifugation data, and Stokes radii obtained by gelfiltration chromatography (Table V). ACP-P1 and ACP-P3 were approximately the same size (130,000 daltons), whereas ACPPz was considerably smaller (108,000 daltons). All three proteins were characterized by a f/fo ratio greater than one, indicating an asymmetric shape. It is noteworthy that theM , 128,000 for ACP-P1, estimated from the combination of data obtained by sucrose density gradient andgel filtration analysis, is inreasonable agreement with the value of 133,000 obtained by the summation of the molecular weights of the two protein bands observed when the most pure preparation of ACP-PI was subjected to polyacrylamide gel electrophoresis in the presence of sodium do-

Phospholipid Enzymes addition

Km

Maximum-fold stimulation

mM

ACP-PI

ACP-P2

ACP-P3

None 1.0 Phosphatidylserine 1.9 Phosphatidylethanolamine

0.35 0.35 0.33

None Phosphatidylserine Monosialoganglioside, 2.6 2.9GM1

2.5

None 1.0 Phosphatidylserine Phosphatidylcholine 3.3

0.44 0.66 0.57

1.7

1.8

1.0 1.8

1.6

20

50

30 40

IO

20

30 5040

Phosphatidylcholine (pg)

Phosphatidylserine (pg)

250

$

300 200 -

-

0 Monosialoganglioside (pg)

IO

20504030

Phosphotadylethanolarnine(pg)

FIG. 7. The effect of various phospholipids on the particulate-bound acid phosphatases of L. donovani. Panels A , E , C, and D show the effect of four phospholipids on L. donovani membrane acid phosphatases. 0, ACP-PI; 0, ACP-P,; and A, ACP-P,. The phospholipids were tested at theconcentrations 2-50 pg/lOO pl in the standard assay mixture.

TABLE V Molecular weight parameters ofL. donovani acid phosphatases Molecular parameters were determined as described under “Experimental Procedures.” The sm,wdata are the average f S.D. of three or more separate analysis. The Stokes radius was calculated from Sephadex G-150 chromatography (1.5 X 115 cm) using blue dextran and NaCl to calculate the void volumeand theretention volume. The column was calibrated with the following standards: 1) lysozyme, 2) ovalbumin, 3) hemoglobin, 4) alkaline phosphatase, and 5) lactate dehvdropenase. Enzyme

ACP-Pi ACP-P2 ACP-P, ~

TABLEIV The effect of different phospholipids onMichaelis constant of membrane-bound acid phosphatases from L. donovani The K,,, values were determined from double-reciprocal plots using MUP as the substrate in the presence of20pgof lipid/standard incubation mixture. The maximum-fold stimulation over control values were calculated from activity values in the presence of 20 pg of lipid/standard incubation mixture.

IO

8

~~

Sz0.u

Molecular Stokes radius

i 1 S.D.

A

6.47 f 0.31 47 5.86 f 0.33 44 7.02 f 0.24 133.000 45 ~

-

weiehts

O ”’

128,000 108,000

1.41 1.39 1.33

~

decyl sulfate (Fig. 3). Substrates-The substrate specificities of the three acid phosphatases derived from the particulate fractionwere compared (Table VI). MUP was the best substrate for all three enzymes, and ACP-P2 and ACP-P3 exhibited broader substrate specificities than ACP-P1. For ACP-P1, the most effective physiologic substrate was fructose 1,6-diphosphate followed by ADP, AMP, and phosphotyrosine. The most effective physiologic substrates for ACP-PZ were fructose 1,6diphosphate, pyridoxal phosphate, and glucose 1-phosphate. Except for serine phosphate, mannose 6-phosphate, mannose 1-phosphate, andpyridoxal phosphate, the substrate specificity of ACP-P3 was similar to that of ACP-P2. Using the sensitive assay for orthophosphate described by Lanzetta et al. (9), we found that the phosphoproteins phosvitin and casein were not substrates for any of the threephosphatases. However, tartrate-resistantACP-PI,the major membrane bound phosphatase, was capable of catalyzing the dephosphorylation of liver pyruvate kinase. The dephosphorylation of

Ps

885

Characterization of L. donovani Acid Phosphatase TABLEVI Substrate specificity of particulate-bound phosphatases of L. donouani All substrates were tested at 1mM concentration except phosvitin (10 mM with respect to phosphate), casein (5 mg/ml), and mannan (10 mg/ml). The values reported below are the result of triplicate determinations. ND, not determined. Substrate ACP-P,

ACP-PI

4-MUP (0.35 100 25.2 0-phospho-DL-tyrosine