Abstract
Renin was completely purified from human kidney cortex employing a rapid three-step procedure which included homogenization and ammonium sulfate precipitation, aminohexyl-pepstatin affinity chromatography, and affinity chromatography using a synthetic octapeptide renin inhibitor (H-77) with a reduced peptide bond (-CH2-NH- instead of -CO-NH-) between Leu5-Leu6, Three kg of cortex dissected from 10 kg of human cadaver kidney yielded 1.7 +/- 0.5 mg of protein (mean +/- S.E. for five procedures) with a specific activity of 1094 +/- 166 Goldblatt units/mg of protein and an overall recovery of 52 +/- 2%. Both gel filtration high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 44,000, although Mr = 22,000 and 18,000 bands were also identified by SDS-PAGE. The pH optima with sheep angiotensinogen were 5.5 and 7.8 and the Km was 0.31 microM. With pure human substrate the pH optimum was 6.0 and the Km was 1.15 microM. Enzyme activity was inhibited by two different anti-human renal renin antibodies. Amino-terminal sequencing demonstrated a leucine residue at the 1-position. Sequencing of 15 additional amino acids agreed with that predicted from the gene sequence and indicated that prorenin is converted to renin following cleavage at the carboxyl end of two basic residues, Lys-2 Arg-1. As with SDS-PAGE analysis, high performance liquid chromatography in the presence of 6 M urea demonstrated Mr = 44,000, 22,000, and 18,000 bands. Immunoblot studies revealed that all of these bands cross-reacted with antihuman renin antibody. Amino-terminal sequencing indicated the Mm = 22,000 band is the amino terminus and the Mr = 18,000 band the carboxyl terminus of Mr = 44,000 renin. In the aqueous phase, these subunits bound to H-77 suggesting that they represent components of the active enzyme complex. Unlike mouse renin, there was no evidence of disulfide bonds. These results raise the question of whether human renin circulates as a subunit aggregation as well as a single chain protein. This may serve as a possible mechanism to regulate renin activity in plasma and tissues.
Highlights
Renin was completely purified from human kidney Interest in the purification of renin (EC 3.4.23.15) stems cortex employing a rapid three-step procedure which from the major role this enzyme plays in the regulation of included homogenization and ammonium sulfate pre- blood pressure
5 N H2S04) andallowed to stand for 16 h a t 4 “C. Following SDS-PAGEno furthershift of the M, = 44,000 renin to smaller species could be detected. These results suggested that humanrenin exists in atleast two forms, one consistingof a single polypeptide chain of M, = 44,000 and the other composed of two chains of lower molecular weight
When the renin containingfractionseluted from the pepstatin-aminohexyl column were subjected to immunoblot analysis, all three bands were present
Summary
To regulate renin activitiyn plasma and tissues. $ Received fellowship support from the American Heart Association, Los Angeles affiliate. To regulate renin activitiyn plasma and tissues. $ Received fellowship support from the American Heart Association, Los Angeles affiliate. 1-3 and 5-8) are presented in miniprint at theend of this paper. Miniprint is read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No 86M-0977,cite the authors, andinclude a check or money order for $4.40 per set of photocopies. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
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