Marquez C, Martinez C, Kroemer G, Bosca L
Centro de Biología Molecular, CSIC, Madrid, Spain.
J Immunol. 1992 Oct 15;149(8):2560-8.
Protein kinase C (PKC) comprises a family of distinct isoenzymes that are involved in signal transduction pathways linking the cell to triggers perceived via membrane receptors. These isoenzymes differ in their tissue distribution, activation requirements, and substrate specificity. One common denominator among different PKC subspecies is their activation by phorbol esters. We have developed a sensitive method permitting the measurement of phorbol ester binding sites, their quantitation, as well as their dissociation kinetics, by performing cytofluorometric analyses on intact cells or on isolated PKC associated to phosphatidylserine vesicles incubated in the presence of fluorochrome-labeled phorbol ester. Both PKC isozymes beta I/beta II and alpha from brain and spleen after incorporation into phosphatidylserine vesicles, display affinities with apparent Kd of 120 and 50 nM, respectively; although PKC gamma from brain exhibits a Kd of 210 nM. In addition to these receptors, on PKC isozymes from spleen, an intermediate affinity phorbol ester receptor (Kd of 3 nM) and an additional high affinity phorbol ester binding site with a Kd of 0.1 to 0.5 nM were also detected. This latter receptor comigrates with high m.w. PKC isoforms. In different cell lines, the phorbol ester binding patterns, as well as the expression of individual PKC isoenzymes, could be positively correlated.
蛋白激酶C（PKC）由一系列不同的同工酶组成，这些同工酶参与将细胞与通过膜受体感知的触发因素相联系的信号转导途径。这些同工酶在组织分布、激活要求和底物特异性方面存在差异。不同PKC亚类的一个共同特征是它们被佛波酯激活。我们开发了一种灵敏的方法，通过对完整细胞或与在荧光染料标记的佛波酯存在下孵育的磷脂酰丝氨酸囊泡相关的分离PKC进行细胞荧光分析，来测量佛波酯结合位点、对其进行定量以及测定其解离动力学。来自脑和脾的PKC同工酶βI/βII和α掺入磷脂酰丝氨酸囊泡后，分别显示出表观Kd为120和50 nM的亲和力；尽管来自脑的PKCγ表现出210 nM的Kd。除了这些受体外，在来自脾的PKC同工酶上，还检测到一个中间亲和力的佛波酯受体（Kd为3 nM）和一个额外的高亲和力佛波酯结合位点，其Kd为0.1至0.5 nM。后一种受体与高分子量的PKC同工型共迁移。在不同的细胞系中，佛波酯结合模式以及各个PKC同工酶的表达可能呈正相关。
