nucleotide-independent protein kinase in Leishmania donovani

1. Leishmania donovani promastigotes labelled for 2 h with 32p, incorporated radioactivity into at least 21 different proteins, as determined by SDS/polyacrylamide-gel electrophoresis. Pulse-chase studies with 32P, demonstrated that the labelled proteins were in a dynamic state: some radiolabelled proteins rapidly disappeared and others appeared after the chase. The possibility of an ectokinase on the parasite was examined; incubation of intact parasites for 10 min at 25 °C in an osmotically buffered medium containing [y-32P]ATP, but not [a-32P]ATP, resulted in the labelling of 10 different protozoal proteins, presumably localized to the surface of the organism's plasma membrane. Intact promastigotes also catalysed the transfer of 32P from [y-32P]ATP to histones. 2. The histone-dependent kinase was solubilized by repeated freezing and thawing, and sonication, and purified 118-fold by chromatographing the high-speed (200000 g, 1 h) supernatant fraction on QAE-Sephadex, Sephadex G-150 and hydroxyapatite columns. The kinase eluted as a single activity peak from all three columns. 3. The partially purified histone-dependent kinase had the following properties: (i) pH optimum, 7.0; (ii) optimum temperature, 37 °C; (iii) Km for mixed calf thymus histone, 0.15 mM; Km for ATP, 0.8 mM; (iv) preferred fractionated histone acceptors, H2b> H4 > H2a > H3 (H1 does not serve as an acceptor); (v) optimum activity required 10-20 mM-Mg2+; (vi) inhibited 50-80% by 0.01 mmand 1 mM-Ca2+; (vii) activity was not stimulated by calmodulin, cyclic AMP (1 mM) or cyclic GMP (1 mM) nor inhibited by a cyclic AMP-dependent protein kinase inhibitor (50 jug/assay); (viii) apparent Mr 75000, as determined by Sephadex G-150 gel filtration chromatography; (ix) phosphorylated exclusively serine residues. 4. Protein kinase activity was low in the early exponential phase of the growth curve and increased 6-fold upon entry into the stationary phase.