Das S, Saha A K, Mukhopadhyay N K, Glew R H
Biochem J. 1986 Dec 15;240(3):641-9. doi: 10.1042/bj2400641.
Leishmania donovani promastigotes labelled for 2 h with 32Pi incorporated radioactivity into at least 21 different proteins, as determined by SDS/polyacrylamide-gel electrophoresis. Pulse-chase studies with 32Pi 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 degrees C in an osmotically buffered medium containing [gamma-32P]ATP, but not [alpha-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 [gamma-32P]ATP to histones. The histone-dependent kinase was solubilized by repeated freezing and thawing, and sonication, and purified 118-fold by chromatographing the high-speed (200,000 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. The partially purified histone-dependent kinase had the following properties: pH optimum, 7.0; optimum temperature, 37 degrees C; Km for mixed calf thymus histone, 0.15 mM; Km for ATP, 0.8 mM; preferred fractionated histone acceptors, H2b greater than H4 greater than H2a greater than H3 (H1 does not serve as an acceptor); optimum activity required 10-20 mM-Mg2+; inhibited 50-80% by 0.01 mM- and 1 mM-Ca2+; 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 micrograms/assay); apparent Mr 75,000, as determined by Sephadex G-150 gel filtration chromatography; phosphorylated exclusively serine residues. Protein kinase activity was low in the early exponential phase of the growth curve and increased 6-fold upon entry into the stationary phase.
用³²P标记杜氏利什曼原虫前鞭毛体2小时后，通过SDS/聚丙烯酰胺凝胶电泳测定，放射性掺入了至少21种不同的蛋白质中。用³²P进行脉冲追踪研究表明，标记的蛋白质处于动态状态：一些放射性标记的蛋白质迅速消失，而另一些在追踪后出现。对寄生虫上存在胞外激酶的可能性进行了研究；完整的寄生虫在含有[γ-³²P]ATP而非[α-³²P]ATP的等渗缓冲培养基中于25℃孵育10分钟，导致10种不同的原生动物蛋白质被标记，推测这些蛋白质定位于生物体质膜表面。完整的前鞭毛体也催化³²P从[γ-³²P]ATP转移到组蛋白上。组蛋白依赖性激酶通过反复冻融和超声处理进行溶解，并通过在QAE-葡聚糖、葡聚糖G-150和羟基磷灰石柱上对高速（200,000g，1小时）上清液部分进行层析而纯化了118倍。该激酶从所有三根柱子上均以单一活性峰洗脱。部分纯化的组蛋白依赖性激酶具有以下特性：最适pH为7.0；最适温度为37℃；混合小牛胸腺组蛋白的Km为0.15mM；ATP的Km为0.8mM；优先的分级组蛋白受体为H2b＞H4＞H2a＞H3（H1不作为受体）；最适活性需要10 - 20mM - Mg²⁺；0.01mM和1mM - Ca²⁺可抑制50 - 80%；钙调蛋白、环磷酸腺苷（1mM）或环磷酸鸟苷（1mM）不刺激其活性，环磷酸腺苷依赖性蛋白激酶抑制剂（50μg/测定）也不抑制其活性；通过葡聚糖G-150凝胶过滤层析测定，表观分子量为75,000；仅磷酸化丝氨酸残基。在生长曲线的早期指数期，蛋白激酶活性较低，进入稳定期后增加6倍。
