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

蛋白激酶C （PKC）是参与许多生理过程信号转导机制的关键酶。这种酶现在被认为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 i, γ, δ， ε和zeta。PKC亚种在酶学特征和细胞位置上表现出微妙的差异，表明它们具有不同的生理作用。我们合成了α、β α、β α和γ - pkc的亚种特异性寡肽，并用这些寡肽免疫家兔获得了亚种特异性抗体。本项目旨在利用电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各亚种的分布有明显差异，与任何一种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、质膜、细胞核、树突和轴突。β - pkc仅位于质膜附近，而β - pkc位于高尔基复合体周围。在海马中，从核周到树突棘，γ - pkc弥漫性分布在锥体细胞的细胞质中。相比之下，β - pkc集中在高尔基复合体周围，并弥散存在于远端树突中，除了树突棘。两个PKC亚种均未在突触末端检测到。这些结果表明，每个PKC亚种在神经元中具有特定的功能，并且γ -PKC和β -PKC参与海马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.