Partial purification and characterization of a soluble protein kinase from Leishmania donovani promastigotes

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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+/phospholipiddid not stimulate enzyme activity. The pH optimum of the enzyme was 7.0-7.5, and the temperature optimum 37.degree. C. The apparent Km for ATP was 60 .mu.M. 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.

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