Partial purification and characterization of the antidiuretic hormone-inactivating enzyme from renal plasma membranes.
An antidiuretic hormone-inactivating peptidase located in renal plasma membranes of porcine kidney medulla has been studied. Treatment of antidiuretic hormone (lysine vasopressin) with renal plasma membranes resulted in a progressive loss of biological activity as measured by the rat pressor assay. The reaction of 2,4,6-trinitrobenzenesulfonic acid with released amino groups was employed to follow the peptidase-catalyzed hydrolysis of the hormone. An 83-fold purification of the membrane-bound peptidase was achieved by Lubrol PX solubilization of the membranes followed by DEAE-cellulose, hydroxylopatite, and 8% agarose column chromatography. The molecular weight of the peptidase was 442 000 as determined by 8% agarose gel filtration. An analysis of the antidiuretic hormone hydrolysis products by thin-layer chromatography revealed the presence of trinitrophenyl-glycinamide. The release of glycinamide from the hormone as a function of time was demonstrated. Mg2+ had a slight inhibitory effect and Ca2+ had a strong inhibitory effect on the peptidase activity.
D000577:Amides; D000818:Animals; D001557:Benzenesulfonates; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D005998:Glycine; D007668:Kidney / Q000201:enzymology*; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009574:Nitro Compounds; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013552:Swine; D014667:Vasopressins
D000577:Amides; D001557:Benzenesulfonates; D009574:Nitro Compounds; D014667:Vasopressins; D010447:Peptide Hydrolases; D008274:Magnesium; D002118:Calcium; D005998:Glycine