Rat angiotensinogen and des(angiotensin I)angiotensinogen: purification, characterization, and partial sequencing

Abstract

Rat angiotensinogen was completely purified by a 6-step procedure including ammonium sulfate precipitation, affinity chromatography on Affi-gel blue, chromatography on DEAE-Sephacel, chromatography on hydroxylapatite, chromatography on Ultrogel AcA 54 and isoelectric focusing. Two peaks of pure angiotensinogen were obtained, distinguishable by their isoelectric points (4.55 and 4.75). Both contained 23 .mu.g of angiotensin I/mg of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the peak with pI = 4.55 revealed 2 protein bands, (respectively, MW 57,000 and 59,000) and a single protein band (MW 57,000) for the peak with pI = 4.75. The MW of the latter homogeneous form, as determined by sedimentation equilibrium was 55,000. Only 1 immunoprecipitin line was observed when antiserum reacted with the heterogeneous angiotensinogen in Ouchterlony gels. The first 17 amino acids of the N-terminal region of the angiotensinogen with pI = 4.75 are reported. The amino acids in positions 10 and 11 which correspond to the renin cleavage site are leucyl-leucyl. The des(angiotensin I)angiotensinogen obtained after hydrolysis of angiotensinogen with pure mouse submaxillary gland renin consisted of a single protein band with an MW of 56,000 as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Only 1 N-terminal residue (leucyl) was obtained for this des(angiotensin I)angiotensinogen. Renin apparently only cleaves angiotensinogen at a single site.