Electron microscopic immunocytochemistry of protein kinase C using subspecies-specific anti-peptide antibody

蛋白激酶C(PKC)是参与多种生理过程的信号转导机制的关键酶。这种酶现在已知是由几个细微不同但密切相关的亚种组成的家族。到目前为止，几个实验室已经分离出编码不同形式的PKC的cDNA克隆，即α、β1、βII、伽马、德尔塔、β和Zeta。PKC亚种在其酶学特性和细胞位置上表现出细微的差异，表明它们具有不同的生理作用。我们合成了针对α、βI、βII和γ-PKC的亚种特异性寡肽，并用这些寡肽免疫兔获得了亚种特异性抗体。本研究的目的是用电子显微镜免疫细胞化学方法阐明神经组织中PKC亚种之间的功能差异。PKC各亚种的分布明显不同，与任何神经递质和受体的分布并不完全一致。在电子显微镜下，γ-PKC存在于核周、质膜、核、树突和轴突中。β-PKC位于质膜附近，而β-PKC位于高尔基复合体周围。在海马区，从核膜到树突棘的锥体细胞胞浆中弥漫分布着γ-PKC。相反，βII-PKC主要集中在高尔基复合体周围，除树突棘外，散在分布于远端树突中。突触终末均未检测到PKC亚种。这些结果表明，每个PKC亚种在神经元中都有特定的功能，伽马-PKC和βII-PKC参与了海马区突触后的长时程增强。

Protein kinase C (PKC) is a key enzyme involved in the signal transduction mechanism of many physiological processes. The enzyme is now known to exist as a family of several subtly different, but closely related subspecies. Thus far several laboratories have isolated cDNA clones encoding distinct forms of PKC, namely, alpha, betaI, betaII, gamma, delta, epsilon, and zeta. PKC subspecies exhibit subtle difference in their enzymological characteristics and cellular locations, suggesting they have distinct physiological roles. We synthesized subspecies-specific oligopeptides for alpha, betaI, betaII, and gamma-PKC and obtained subspecies-specific antibodies by immunizing rabbits with the oligopeptides. We aimed in this project to elucidate the functional difference between the subspecies of PKC in nervous tissue using electron microscopic immunocytochemistry. The distribution of each PKC subspecies was distinctly different and the distributions did not completely correspond with those of any neurotransmitters and receptors. Under electron microscopy, gamma-PKC was present in the perikaryon, plasma membrane, nucleus, dendrite, and axon. betaI-PKC was just adjacent to plasma membrane, while betaII-PKC was located around Golgi complex. In the hippocampus, gamma-PKC was distributed diffusely throughout the cytoplasm of pyramidal cells from the perikarya to dendritic spine. In contrast, betaII-PKC was concentrated around Golgi complex and present diffusely in distal dendrites, except for the dendritic spines. Neither PKC subspecies could not be detected in the synaptic terminals. These results suggest that each PKC subspecies has a specific function in the neurons and that gamma-PKC and betaII-PKC are involved in long term potentiation postsynaptically in the hippocampus.