Activity Staining Method of Phosphorylase by Polyacrylamide Gel Disc Electrophoresis and Demonstration of Phosphorylase lsozymes
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By using polyacrylamide gel disc electrophoresis, we developed a simple and sensitive stain method for glycogen phosphorylase (EC 2. 4. 1. 1). The method is highly sensitive for demonstration of phosphorylase activity which could not be detected by any conventional method.<BR>In view of electrophoretic mobility, rabbit tissue phosphorylases could be classified into two groups: the faster moving ones of brain and kidney, and the slower moving ones of skeletal muscle and liver. With electrophoresis gel containing glycogen to a considerable concentration, the mobilities were retarded. The retardation was more marked for the muscle and the brain phosphorylases than for those of liver and kidney. Therefore, all four tissue phosphorylases might be organ-specific.<BR>Retardation of mobility of phosphorylase fraction was increased in proportion to glycogen concentration in the gels. On the other hand, in regard to protein fractions other than phosphorylase, no retardation was observed. On the basis of variation in mobility as a function of glycogen concentration, the dissociaiton constant of phosphorylase with glycogen could be calculated. The values obtained were 6.6×10<SUP>-4</SUP> M, 24×10<SUP>-4</SUP>M, and 13×10<SUP>-4</SUP>M for phosphorylases of rabbit skeletal muscle, liver and brain, respectively.<BR>In some pathological conditions, phosphorylase activity was demonstrated in human urines. In the extract of human kidney, two phosphorylase fractions were demonstrated. In the urines of hydronephrosis associated with renal calculi, of unilateral renal tuberculosis, and of ureteral carcinoma, one phosphorylase activity was demonstrated in the position, corresponding to the slower moving fraction of kidney phosphorylase. In the urine of plumonary carcinoma, another phosphorylase fraction was demonstrated at the position a little faster than the former. It corres-ponded to the faster moving fraction of kidney phosphorylase. In regard to ureteral carcinoma, the catheterized urine, which was collected 2 weeks after surgical removal of the affected kidney and tissues, showed phosphorylase activity. In contrast, the urine of renal tuberculosis showed no phosphorylase activity after removal of the affected kidney.
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- Activity Staining Method of Phosphorylase by Polyacrylamide Gel Disc Electrophoresis and Demonstration of Phosphorylase lsozymes