Mosior M, Newton A C
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0640, USA.
J Biol Chem. 1995 Oct 27;270(43):25526-33. doi: 10.1074/jbc.270.43.25526.
A variety of approaches have been employed to demonstrate that the interaction of protein kinase C beta II with phorbol ester-containing membranes is reversible, is not accompanied by significant insertion of the protein into the hydrophobic core of the membrane, and is qualitatively similar to the interaction with diacylglycerol (DG). First, we show that under conditions when protein kinase C is bound with equal affinity to membranes containing either DG or phorbol myristate acetate (PMA), increasing ionic strength causes a similar reduction in membrane binding. The similar sensitivity to ionic strength indicates that the forces mediating the binding of protein kinase C to PMA are not significantly different from those mediating the binding to DG. At sufficiently high concentrations of PMA and relatively low concentrations of phosphatidylserine, the binding of protein kinase C to membranes became markedly less sensitive to ionic strength, suggesting that under these conditions direct non-electrostatic interactions with PMA dominate over electrostatic interactions with the lipid headgroups. Importantly, regardless of the strength of the interaction with PMA, protein kinase C exchanges between vesicle surfaces: protein kinase C bound first to phorbol ester-containing multilamellar vesicles exchanged to large unilamellar vesicles upon addition of an excess surface area of the latter. Lastly, the enzyme's intrinsic tryptophan fluorescence was not quenched by bromines located at various positions in the hydrophobic core of the membrane. In contrast, the enzyme's tryptophan fluorescence was significantly quenched by probes positioned at the membrane surface. In summary, our results are consistent with protein kinase C binding reversibly to PMA- or DG-containing membranes primarily via interactions at the membrane interface.
人们采用了多种方法来证明蛋白激酶CβII与含佛波酯的膜之间的相互作用是可逆的，该蛋白不会大量插入膜的疏水核心，并且在性质上与和二酰基甘油（DG）的相互作用相似。首先，我们发现，在蛋白激酶C以相同亲和力结合含有DG或佛波酯肉豆蔻酸酯（PMA）的膜的条件下，增加离子强度会导致膜结合能力出现类似程度的降低。对离子强度的相似敏感性表明，介导蛋白激酶C与PMA结合的力与介导其与DG结合的力没有显著差异。在足够高浓度的PMA和相对低浓度的磷脂酰丝氨酸条件下，蛋白激酶C与膜的结合对离子强度的敏感性明显降低，这表明在这些条件下，与PMA的直接非静电相互作用比与脂质头部基团的静电相互作用更为重要。重要的是，无论与PMA相互作用的强度如何，蛋白激酶C都会在囊泡表面之间交换：首先结合到含佛波酯的多层囊泡上的蛋白激酶C在加入过量的大单层囊泡后会交换到后者上。最后，膜疏水核心中不同位置的溴不会淬灭该酶固有的色氨酸荧光。相反，位于膜表面的探针会显著淬灭该酶的色氨酸荧光。总之，我们的结果与蛋白激酶C主要通过在膜界面的相互作用而可逆地结合到含PMA或DG的膜上这一观点一致。
