Rapid and large-scale purification and characterization of renin from mouse submaxillary gland

Abstract

Little is known about the structural basis for the highly restricted substrate specificity of renin, whose only function known to date is to hydrolyze the unique leucyl peptide bond in the prohormone angiotensinogen to form angiotensin I. Our lack of knowledge is due to our inability to purify this enzyme in a large quantity sufficient for structural studies. A two-step column chromatographic method for rapid and large-scale purification of renin from mouse submaxillary gland has been developed. It allows isolation of the enzyme in a hundreds-of-milligrams quantity at an overall yield of 60%. Single bands obtained by polyacrylamide gel electrophoresis, isoelectric focusing, and the result of ultracentrifugal studies indicated homogeneity of the product. The moelcular weight of renin was estimated to be 36,000 by sedimentation equilibrium studies in 6 m guanidine · HCl. Sedimentation velocity study gave a single sedimenting boundary with an s 20,w of 2.58 × 10 −13 s. A Stokes radius of 27 Å was obtained by gel filtration. The far ultraviolet-circular dichroism spectrum indicated a high content of β-structure (46%). In contrast to renal renin, submaxillary gland renin does not contain amino sugars or neutral sugars. No renin activity was retained by concanavalin Aagarose gels. Results of amino acid analysis, isoelectric focusing, and determination of amino-terminal residues by the dansylchloride reaction, together indicated that this protein is identical with renin A isolated previously in a much smaller quantity.