Distribution of mu-calpain proenzyme in the brain and other neural tissues in the rat.
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.
Onizuka
K
K
Department of Physiology, Nagoya City University Medical School, Japan.
Kunimatsu
M
M
Ozaki
Y
Y
Muramatsu
K
K
Sasaki
M
M
Nishino
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000918:Antibody Specificity; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D010525:Peripheral Nerves / Q000201:enzymology*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar
D004792:Enzyme Precursors; D010446:Peptide Fragments; D002154:Calpain; C098484:mu-calpain proenzyme