Application of immunochemical methods to the identification and characterization of rat kidney inactive renin.
Identification of inactive prorenin in the kidney has been difficult due to rapid proteolytic conversion of the inactive zymogen to its active form in the tissue or during homogenization and purification. Immunochemical methods, Western blotting, direct radioimmunoassay, and immunoaffinity chromatography were used to isolate and identify rat kidney renin and prorenin and to determine their molecular weights without complete purification. Antisera to pure rat renin were raised in rabbits. A specific reaction between the antisera and rat renin was demonstrated by double immunodiffusion, inhibition of enzyme activity, and competitive radioimmunoassay. The anti-rat renin IgG did not cross-react with purified human renin or rat spleen or kidney cathepsin D. The IgG showed binding affinity to both inactive renin as well as active enzyme. A combination of affinity chromatographies consisting of pepstatin-Sepharose, IgG-Sepharose, and Affi-Gel Blue permitted rapid and complete separation of inactive renin from active renin in rat kidney extract. Neither inactive nor active renin preparations exhibited aspartyl protease activity on hemoglobin used as substrate. The apparent molecular weight of inactive renin was estimated as 50,000 by gel filtration. Electrophoresis of partially purified inactive renin in sodium dodecyl sulfate (SDS) polyacrylamide gel followed by transblotting of proteins to a nitrocellulose sheet and immunochemical staining with anti-renin IgG showed a single protein band with a molecular weight of 48,000. Activation of inactive renin by trypsin was accompanied by the reduction of the 48,000-dalton native protein to a 39,000-dalton protein as determined by the SDS polyacrylamide gel electrophoresis and the transblotting.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002402:Cathepsin D / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007668:Kidney / Q000201:enzymology* / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D011863:Radioimmunoassay; D051381:Rats; D012083:Renin / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D013154:Spleen / Q000378:metabolism
D000906:Antibodies; D004792:Enzyme Precursors; D007074:Immunoglobulin G; D012083:Renin; D002402:Cathepsin D