title: Partial purification and characterization of a soluble protein kinase from Leishmania donovani promastigotes.
abstract: A soluble protein kinase from the promastigote form of the parasitic protozoon Leishmania donovani was partially purified using DEAE-cellulose, Sephadex G-200 and phosphocellulose columns. The enzyme preferentially utilized protamine as exogenous phosphate acceptor. The native molecular mass of the enzyme was about 85 kDa. Mg2+ ions were essential for enzyme activity; other metal ions, e.g. Ca2+, Co2+, Zn2+ and Mn2+, could not substitute for Mg2+. cAMP, cGMP, Ca2+/calmodulin and Ca2+/phospholipid did not stimulate enzyme activity. The pH optimum of the enzyme was 7.0-7.5, and the temperature optimum 37 degrees C. The apparent Km for ATP was 60 microM. Phosphoamino acid analysis revealed that the protein kinase transferred the gamma-phosphate of ATP to serine residues in protamine. The thiol reagents p-hydroxymercuribenzoic acid, 5-5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide inhibited enzyme activity; the inhibition by p-hydroxymercuribenzoic acid and 5-5'-dithio-bis(2-nitrobenzoic acid) was reversed by dithiothreitol.
authors.affiliation: Leishmania Group, Indian Institute of Chemical Biology, Jadavpur, Calcutta.
authors.forename: C
authors.initials: C
authors.lastname: Banerjee
authors.forename: D
authors.initials: D
authors.lastname: Sarkar
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002845:Chromatography; D003035:Cobalt / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013696:Temperature; D015032:Zinc / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D003035:Cobalt; D008345:Manganese; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D008274:Magnesium; D015032:Zinc; D002118:Calcium