Purification, of Rat Liver Mitochondrial Ornithine Carbamyoltransferase and Properties of Galactosamine:treated Rat Serum Ornithine Carbamoyltransferase
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Ornithine carbamoyltransferase (OCT) is almost exclusively located in the liver mitochondria'matrix of ureoteric animals. A trace activity of OCT in normal serum has been found and the enzyme activity increased markedly in patients with acute hepatic diseases. Therefore, those phenomena have been used as an index for diagnosing some hepatic diseases, although few studies have been made on the properties of the enzyme in serum, and there is no direct evidence whether the serum enzyme was released from the matrix of liver mitochondria.<BR>Here I report that OCT has been homogeneously purified by ammonium sulfate fractionation, heat treatment,2nd ammonium sulfate fractionation, Phenyl Sepharose CL-4B and CM-Sephadex C-50 column chromatographies. The purified enzyme exhibited the molecular weight of 39,500as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and of 115,000 by gel filtration. In addition, the characteristics of serum OCT in galactosaminetreated rats and variations in the amount and the activity of the serum enzyme after treatment with galactosamine were investigated. A single injection of D-galactosamine (90mg/100g of body weight) into rats caused acute liver cell injury, and the activity of OCT in the serum increased about 600 times as compared with that in normal serum. The serum and mitochondrial enzyme activities gave the similar pH profiles, showing an optimum of pH 8.5. Apparent Km values of the serum enzyme for ornithine under the assay conditions at pH 7.4 and 7.7 were 1.59mM and 0.94mM, respectively, and those of the mitochondria' enzyme for the corresponding substrate were 1.69 mM and 0.97 mM, respectively. The Km value of the serum enzyme for carbamyl phosphate was 0.34 mM, which was similar to that of the mitochondrial enzym e. The mitochondrial and serum enzymes had a molecular weight of 115,000 by Sephacryl S-300gel filtration. The antibody against the mitochondria' enzyme was raised, and the immunological properties of the serum enzyme were examined. The experiments of immunoinhibition showed that an increase in the antibody caused both the serum enzyme activity and the mitochondrial enzyme activity similarly to decrease. Analysis by SDS-PAGE showed that the molecular weights of the serum and mitochondrial enzymes were identical. The amounts of the enzyme varied in a manner similar to the variations in the activity. Those results indicated that OCT was released into the serum in an intact form from the mitochondrial matrix during hepatic damage.
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