Characterization of Leishmania donovani acid phosphatases.
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 (200,000 X 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 A-Sepharose, QAE-Sephadex, and Sephadex G-150 and G-100. Three distinct acid phosphatases were resolved: the major phosphatase activity (70% of the total) was L-(+)-tartrate-resistant (designated ACP-P1) and corresponds to the acid phosphatase localized to the outer surface of the parasite's plasma membrane; the other two phosphatases (ACP-P2 and ACP-P3) account for the remaining 30% of the particulate acid phosphatase activity, and both 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 determined that ACP-P1 is a 128,000-dalton protein composed of two subunits of 65,000-68,000 daltons. ACP-P1 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 pyruvate kinase. ACP-P2 (pI, 5.4) and ACP-P3 (pI, 7.1) with molecular masses of 132,000 and 108,000 daltons, respectively, are both tartrate-sensitive and are distinguished from each other on the basis of their sensitivity to inhibition by polyanionic molybdenum complexes. These two phosphatases also have their pH optima in the pH 5.0-6.0 range, but have a considerably broader substrate specificity than ACP-P1.
D000135:Acid Phosphatase / Q000378:metabolism*; D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006859:Hydrogen; D007526:Isoelectric Point; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D007891:Leishmania / Q000201:enzymology*; D008055:Lipids / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008970:Molecular Weight
D007527:Isoenzymes; D008055:Lipids; D015636:Magnesium Chloride; D006859:Hydrogen; D000135:Acid Phosphatase; D008274:Magnesium; D002122:Calcium Chloride