Production and characterization of an antibody to cytosolic aspartate aminotransferase and immunolocalization of the enzyme in rat organs.
Cytosolic aspartate aminotransferase (c-AAT) was purified to homogeneity from porcine heart and immunized to rabbit for production of antiserum. The purity of this enzyme protein and the specificity of its antibody were judged by silver-stained-sodium dodecyl sulfate slab gel, Western blot transfer technique, and double immunodiffusion. The antibody against porcine heart c-AAT was found to cross-react with rat c-AAT but not with nine other different enzymes from the heart, liver, and muscle. Affinity purified antibody was used to localize this isoenzyme in the rat heart, liver, kidney, and cerebellum by indirect immunoperoxidase method. It was found that, in the rat heart muscle, c-AAT reaction product was present as a linear structure parallel to the muscle fiber and along the sarcolemma. Some cardiac muscle fibers contain more reaction products than the others. In the liver, reaction product was seen unevenly distributed in the hepatocytes. The Kupffer cells and endothelia were less stained. Most of the tubular epithelia of the loop of Henle in the kidney were intensely stained. But other tubular epithelia including convoluted and collecting tubules were sporadically and less stained. The basket and stellate cells and their neuronal processes and terminals in the cerebellum were markedly stained, but the Purkinje and granule cell bodies were weakly stained. For comparison of the staining intensity with enzyme activity in each organ, the c-AAT enzyme activity was simultaneously determined in those organs. This study indicates that the presence of c-AAT is specific in different organs and tissues.
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001219:Aspartate Aminotransferases / Q000276:immunology*; D002531:Cerebellum / Q000201:enzymology; D006801:Humans; D005779:Immunodiffusion; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008854:Microscopy, Electron; D009206:Myocardium / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D013552:Swine; D014018:Tissue Distribution
D000906:Antibodies; D001219:Aspartate Aminotransferases