Bazzi M D, Nelsestuen G L
Biochemistry. 1987 Apr 7;26(7):1974-82. doi: 10.1021/bi00381a029.
The role of substrate in influencing the cofactor requirements of the phospholipid- and Ca2+-dependent protein kinase C (PKC) was investigated by using several substrates. All of the substrates tested, including histone, troponin I, myosin light chain, protamine, poly(arginine, serine) (PAS), poly(lysine, serine) (PLS), and myelin basic protein (MBP), were found to interact with and aggregate phospholipid vesicles as well as phosphatidylserine (PS)-Triton mixed micelles. Phosphorylation of these different substrates by PKC indicated the presence of three distinct substrate categories: substrates such as protamine requiring no cofactors; substrates such as PLS, PAS, and MBP requiring only the presence of phospholipid; and substrates such as histone, myosin light chain, and troponin I requiring the presence of Ca2+ and phospholipid. Diacylglycerol was a major cofactor only with category C substrates. These different requirements correlated with the interaction of the substrate with phospholipid and/or enzyme. The substrates in category A interacted strongly with and aggregated PKC in a binary mixture. In the absence of Ca2+, PKC bound to substrates of category B directly but not to substrates in category C. Thus, substrate-enzyme binding eliminated the Ca2+ requirement of phosphorylation, and aggregation of substrate-enzyme complex eliminated the phospholipid requirements as well. Substrate-phospholipid interaction and substrate phosphorylation were inhibited by increasing salt concentrations, but the amount needed depended upon the substrate. Loss of PKC activity appeared to coincide with loss of substrate-PS aggregation while dissociation of PKC from the membranes required much higher salt concentrations. Poly(L-lysine) and poly(L-arginine), two potent inhibitors of PKC, also showed substrate-dependent inhibition characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)
通过使用多种底物，研究了底物在影响磷脂和钙离子依赖性蛋白激酶C（PKC）辅助因子需求方面的作用。所测试的所有底物，包括组蛋白、肌钙蛋白I、肌球蛋白轻链、鱼精蛋白、聚（精氨酸，丝氨酸）（PAS）、聚（赖氨酸，丝氨酸）（PLS）和髓鞘碱性蛋白（MBP），均被发现可与磷脂囊泡以及磷脂酰丝氨酸（PS）-曲拉通混合胶束相互作用并使其聚集。PKC对这些不同底物的磷酸化表明存在三种不同的底物类别：如鱼精蛋白等不需要辅助因子的底物；如PLS、PAS和MBP等仅需要磷脂存在的底物；以及如组蛋白、肌球蛋白轻链和肌钙蛋白I等需要钙离子和磷脂存在的底物。二酰基甘油仅是C类底物的主要辅助因子。这些不同的需求与底物与磷脂和/或酶的相互作用相关。A类底物在二元混合物中与PKC强烈相互作用并使其聚集。在没有钙离子的情况下，PKC直接与B类底物结合，但不与C类底物结合。因此，底物-酶结合消除了磷酸化对钙离子的需求，底物-酶复合物的聚集也消除了对磷脂的需求。增加盐浓度会抑制底物-磷脂相互作用和底物磷酸化，但所需的量取决于底物。PKC活性的丧失似乎与底物-PS聚集的丧失同时发生，而PKC从膜上解离则需要更高的盐浓度。聚（L-赖氨酸）和聚（L-精氨酸）这两种PKC的强效抑制剂也表现出底物依赖性抑制特性。（摘要截短于250字）
