Biochemical studies on the possible mechanisms of action of vasopressin on canine renal medulla



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The possible subcellular mechanisms of Vasopressin action in the canine renal medulla were studied. Using the techniques of zonal density gradient ultracentrifugation and free flow electrophoresis, the apical and basolateral membranes of the canine renal medulla were purified. Na[superscript 3]-K[superscript 3]ATPase, Vasopressin stimulated adenylate cyclase and [superscript 3]H- Vasopressin binding sites were used as markers for the basolateral membranes and showed thirty, twenty and thirty-three fold enrichment in terms of specific activities respectively in the purified membrane fraction. HCO[subscript 3]-ATPase was used as a marker for the apical membrane and showed a twenty-nine fold enrichment in terms of specific activity in the purified apical membrane fraction. This enzyme could be distinguished from the mitochondrial HCO[subscript 3]ATPase due to its resistance to inhibition by the protein inhibitor for mitochondrial ATPases. The apical membranes but not the basolateral membranes were found to contain a membrane bound cyclic AMP dependent protein kinase, and a phosphoprotein phosphatase. The substrate was a single class of polypetide(s) with an apparent molecular weight of 46,000 as determinedby SDS-polyacrylamide gel electrophoresis. Maximal stimulation of protein Kinase was observed at 1[mu]M cyclic AMP. Km for ATP in the presence of 1[mu]M cyclic AMP, was 5[mu]M.