Purification of high molecular weight (HMW) renin from porcine kidney and direct evidence that the HMW renin is a complex of renin with renin binding protein (RnBP).
The high molecular weight (HMW) renin was purified from porcine kidney by a procedure involving extraction with a buffer system containing protease inhibitors, ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B column chromatography, gel filtration on Ultrogel AcA 44 and aminohexyl-Sepharose 4B column chromatography. The resulting preparation showed a single band on isoelectric focusing, exhibiting an isoelectric point at pH 5.25, and was stable on storage at -80 degrees C for 4 months. The specific activity was 3.97 mg of angiotensin I formed/mg of protein per h at 37 degrees C and at pH 6.5 with porcine angiotensinogen as the substrate. When the HMW renin was exposed to acid, renin activity increased by about 5-fold and the free form of fully active renin was recovered from the acidified HMW renin, leaving an insoluble aggregate of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the HMW renin showed two protein bands, of which one was identified as renin from the electrophoretic mobility and the other was the protein, assigned as renin binding protein (RnBP), that was insolubilized by acidification. The purified HMW renin is a complex of renin with RnBP, and the molecular weights of RnBP and renin in the HMW renin were estimated to be 39,000 and 32,000, respectively, by gel permeation liquid chromatography in 6 M guanidine-HCl. A modified rapid method for purification of renin is also presented.
D000818:Animals; D002238:Carbohydrate Epimerases; D002352:Carrier Proteins / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000032:analysis*; D008970:Molecular Weight; D012083:Renin / Q000302:isolation & purification*; D013552:Swine
D002352:Carrier Proteins; D012083:Renin; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase