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信号转导的关键酶。现在已知这种酶是一个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PKC的cDNA克隆，即α、β I、β II、γ、δ、β 1和ζ。PKC亚型在酶学特性和细胞定位上存在细微差异，提示它们具有不同的生理功能。我们合成了α，β I，β II和γ-PKC的亚种特异性寡肽，并通过用寡肽免疫家兔获得亚种特异性抗体。本课题旨在通过电镜免疫细胞化学技术研究PKC在神经组织中不同亚型间的功能差异。PKC各亚型的分布明显不同，与神经递质和受体的分布不完全一致。电镜下，γ-PKC存在于核周体、质膜、细胞核、树突和轴突中。β I-PKC位于质膜附近，而β II-PKC位于高尔基复合体周围。在海马，γ-PKC弥漫分布在整个细胞质的锥体细胞从胞体树突棘。相反，β II-PKC集中在高尔基复合体周围，并弥漫存在于远端树突，除了树突棘。在突触终末中均未检测到PKC亚型。这些结果表明，每个PKC亚型在神经元中具有特定的功能，并且γ-PKC和β II-PKC参与海马突触后长时程增强。

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.