Phosphatidylglycerol-modulated protein kinase activity from human spleen. I. Enzyme purification and properties.
Phosphatidylglycerol-modulated protein kinase activity from human spleen. I. Enzyme purification and properties.
复制标题
来自人脾脏的磷脂酰甘油调节的蛋白激酶活性。
DOI：
10.1016/0003-9861(88)90154-3
复制
发表时间：
1988
影响因子：
3.9
通讯作者：
Elias,L
中科院分区：
文献类型：
--
作者：
Klemm,DJ;Kazim,AL;Elias,L
蛋白激酶P （PK-P）是一种磷脂调节的蛋白激酶活性，先前在人类和小鼠细胞中被描述过。本文详细介绍了用三步色谱法纯化人脾蛋白激酶P的3300倍高产量电泳均匀性。物理表征揭示了mr27,000（电泳）或31,700（凝胶过滤和沉淀）和ptI5.09的蛋白。磷脂酰甘油或磷脂酰肌醇均能刺激蛋白激酶P活性，磷脂浓度在200 ~ 400 μg/ml之间刺激最大。使用磷脂酸或磷脂酰丝氨酸无刺激作用。组蛋白H2B是显示蛋白激酶P磷脂刺激的最佳底物。组蛋白h1以不依赖磷脂的方式磷酸化。血管蛋白和肌动蛋白不是底物。在35°C和pH 6.95的条件下，酶活性达到最佳。PK-P对钙调素和蛋白激酶C抑制剂w7和H7以及camp依赖性蛋白激酶抑制剂相对不敏感。动力学分析揭示了复杂的模式，包括组蛋白和磷脂浓度的最佳动力学而不是Michaelis-Menton动力学，以及磷脂存在时组蛋白浓度的陡峭激活阈值。观察到Mg2+-ATP的双相动力学，磷脂的主要刺激作用是在vmax而不是km上。这些数据表明，磷脂激活蛋白激酶P的机制模型涉及底物、酶和磷脂胶束之间的直接三方相互作用，而不是磷脂的变构激活。
Protein kinase P (PK-P) is a phospholipid-modulated protein kinase activity previously described in human and murine cells. This paper details the 3300-fold, high yield purification to electrophoretic homogeneity of protein kinase P from human spleen by a three-step chromatographic process. Physical characterization disclosed a protein ofMr27,000 (by electrophoresis) or 31,700 (by gel filtration and sedimentation) and ptI5.09. Protein kinase P activity was stimulated by phosphatidylglycerol or phosphatidylinositol, with maximal stimulation observed between 200 and 400 μg/ml phospholipid. No stimulation was noted using phosphatidic acid or phosphatidylserine. Histone H2B was the best substrate for demonstrating the protein kinase P phospholipid stimulation. Histone H1was phosphorylated in a phospholipid independent manner. Vinculin and actin were not substrates. Optimum enzyme activity was observed at approximately 35 °C and pH 6.95. PK-P was relatively insensitive to the calmodulin and protein kinase C inhibitors W7and H7, and to the cAMP-dependent protein kinase inhibitor. Kinetic analysis disclosed complex patterns including optimal rather than Michaelis-Menton kinetics for histone and phospholipid concentration, and a steep activation threshold with respect to histone concentration in the presence of phospholipid. Biphasic kinetics for Mg2+-ATP were observed, with the major stimulatory effect of phospholipid being onVmaxrather thanKm. These data suggest a model for the mechanism of activation of protein kinase P by phospholipid entailing a direct three-way interaction between substrate, enzyme, and phospholipid micelles rather than allosteric activation by phospholipid.