Onizuka K, Kunimatsu M, Ozaki Y, Muramatsu K, Sasaki M, Nishino H
Department of Physiology, Nagoya City University Medical School, Japan.
Brain Res. 1995 Oct 30;697(1-2):179-86. doi: 10.1016/0006-8993(95)00838-h.
We raised antibodies against the acetyl N-terminal peptide of the human mu-calpain 80 kDa (80 K) subunit (N-acetyl SEETPVYCT-GVSAQVQKQRARELG) in the rabbit. A specific antibody was purified using N-acetyl SEEITPVYCTGVSAQVQKQ peptide-conjugated Sepharose 4B as an affinity gel support. Epitope analysis revealed that the purified antibody reacted only with mu-calpain N-terminal peptides containing N-acetyl SEETT structure but no reactions occurred with other analogous peptides. Western blot analysis showed that the antibody reacted with both human and rat mu-calpain proenzymes but not with the activated calpains lacking N-terminal peptide. Using this antibody we investigated immunohistochemically the distribution of mu-calpain proenzyme in central and peripheral nervous systems as well as other non-neural tissues in the rat. The proenzyme was detected mainly in neurons both in the central and peripheral nervous tissues, but not in non-neural tissues except for red blood cells. Immunoreaction was stronger in the perikarya and/or in the nuclei than in-the cytoplasm. Specificity of the antibody was verified by an absorption test. In summary, the mu-calpain proenzyme is mainly distributed in the perikarya and/or nuclei or neurons. Our present antibody specific to the N-terminus of the mu-calpain 80 K subunit could serve as a useful tool to detect various functions of mu-calpain as well as the damage in neurons caused by the enzyme.
我们在兔体内制备了针对人μ-钙蛋白酶80 kDa（80K）亚基乙酰化N端肽（N-乙酰基SEETPVYCT-GVSAQVQKQRARELG）的抗体。使用N-乙酰基SEEITPVYCTGVSAQVQKQ肽偶联的琼脂糖4B作为亲和凝胶支持物纯化特异性抗体。表位分析表明，纯化的抗体仅与含有N-乙酰基SEETT结构的μ-钙蛋白酶N端肽发生反应，而与其他类似肽无反应。蛋白质印迹分析表明，该抗体与人及大鼠的μ-钙蛋白酶原酶均发生反应，但与缺乏N端肽的活化钙蛋白酶无反应。利用该抗体，我们通过免疫组织化学方法研究了大鼠中枢和外周神经系统以及其他非神经组织中μ-钙蛋白酶原酶的分布。在中枢和外周神经组织中，原酶主要在神经元中检测到，但在除红细胞外的非神经组织中未检测到。免疫反应在核周体和/或细胞核中比在细胞质中更强。通过吸收试验验证了抗体的特异性。总之，μ-钙蛋白酶原酶主要分布在核周体和/或细胞核或神经元中。我们目前针对μ-钙蛋白酶80K亚基N端的特异性抗体可作为检测μ-钙蛋白酶各种功能以及该酶引起的神经元损伤的有用工具。
