Chan K F
Laboratory of Experimental Neuropathology, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20892.
J Biol Chem. 1988 Jan 5;263(1):568-74.
A novel protein kinase which could be inhibited specifically by gangliosides has been partially purified from the particulate fraction of guinea pig brain through extraction with nonionic detergent, ion-exchange chromatography, hydrophobic chromatography, hydroxylapatite chromatography, and gel filtration. The ganglioside-inhibited kinase activity was eluted with a Stokes radius of 29-30 A, corresponding to a globular protein of approximately 40,000 in molecular weight. Only gangliosides, especially polysialogangliosides, are potent inhibitors for this enzyme preparation. The modulatory action of the glycolipids on the kinase activity is not time-dependent, indicating that the mode of inhibition may not be mediated through a ganglioside-dependent proteolytic process. Calcium was not required for the inhibitory effects of the various gangliosides tested, suggesting that prior formation of Ca2+.ganglioside complexes are not necessary. The partially purified ganglioside-inhibited protein kinase can phosphorylate exogenous substrates such as a synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly. The optimal pH for this reaction occurred between 7.0 and 7.4. Mg2+ (5-10 mM) is required for the enzymic activity and cannot be substituted by Mn2+. Although the nature of the authentic substrates for this ganglioside-inhibited protein kinase is yet unknown, a search for other potential substrates revealed that the synthetic peptide Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val was the best phosphate acceptor tested so far. Other substrate specificity studies also showed that the ganglioside-inhibited protein kinase is distinct from either the ganglioside-stimulated protein kinase or protein kinase C. Thus, it is possible that gangliosides can act as bio-modulators which may confer a synchronistic action on these three different protein kinase systems.
一种可被神经节苷脂特异性抑制的新型蛋白激酶已通过用非离子去污剂提取、离子交换色谱、疏水色谱、羟基磷灰石色谱和凝胶过滤，从豚鼠脑的微粒部分中部分纯化出来。神经节苷脂抑制的激酶活性以斯托克斯半径29 - 30 Å洗脱，对应于分子量约为40,000的球状蛋白。只有神经节苷脂，尤其是多唾液酸神经节苷脂，是这种酶制剂的有效抑制剂。糖脂对激酶活性的调节作用不依赖时间，这表明抑制模式可能不是通过神经节苷脂依赖性蛋白水解过程介导的。测试的各种神经节苷脂的抑制作用不需要钙，这表明Ca2⁺·神经节苷脂复合物的预先形成不是必需的。部分纯化的神经节苷脂抑制的蛋白激酶可以磷酸化外源性底物，如合成肽Leu - Arg - Arg - Ala - Ser - Leu - Gly。该反应的最佳pH值在7.0至7.4之间。酶活性需要Mg2⁺（5 - 10 mM），且不能被Mn2⁺替代。尽管这种神经节苷脂抑制的蛋白激酶的真实底物的性质尚不清楚，但对其他潜在底物的搜索显示，合成肽Arg - Arg - Lys - Ala - Ser - Gly - Pro - Pro - Val是迄今为止测试的最佳磷酸受体。其他底物特异性研究还表明，神经节苷脂抑制的蛋白激酶与神经节苷脂刺激的蛋白激酶或蛋白激酶C不同。因此，神经节苷脂有可能作为生物调节剂，对这三种不同的蛋白激酶系统产生同步作用。
