Partial purification and characterization of particulate acid phosphatase of Leishmania donovani promastigotes.

1. More than 90% of the total acid phosphatase activity in a sonicate of L. donovani promastigotes is contained in a particulate fraction (200,000 X g 30 min). The enzyme can be quantitatively extracted and solubilized with the aid of Triton X-100 (0.2 g/100 ml) and purified over 200-fold with 54% yield by chromatography on DEAE-Sephadex, QAE-Sephadex, Sepharose 4B and concanavalin-A Sepharose. 2. The phosphatase is a true acid hydrolase (pH optimum, 5.0-5.5) and has a rather broad substrate specificity; it will catalyze the hydrolysis of 4-methylumbelliferylphosphate, thymolphthalein diphosphate, pyridoxal phosphate, fructose 1,6-diphosphate, glucose 6-phosphate, glucose 1-phosphate, ADP and AMP. 3. It is a large (170,000 daltons in the presence of Triton X-100), stable and acidic enzyme (pI = 4.1) that has the electrophoretic mobility of a type zero or type 1 isoenzyme in acid (pH 4.3) polyacrylamide gels. 4. The enzyme is inhibited by sodium fluoride, 2-mercaptoethanol and mumolar amounts of a number of polyanionic molybdenum and heavy metal complexes that include the following: [C(NH2)3]4[(C3H7O3PO3)2Mo5O15] X 3H2O, [C(NH2)3]2[(C6H5)2AsMo4O15H] X H2O, (NH4)4[SiMo12O40] X H2O and (NH4)6[P2Mo18O62] X 9H2O. 5. L. donovani promastigotes contain very low levels of 10 other acid pH optimum hydrolytic enzymes, with the exception of modest levels of alpha-fucosidase.