Purification and characterization of two forms of rat plasma proangiotensin.
Two forms of rat plasma proangiotensin were purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose at pH 6.5, DEAE-Sepharose at pH 8.9, Sephadex G-150, hydroxyapatite and hexyl-agarose. Both forms were finally separated by affinity chromatography on concanavalin-A--Sepharose. Presence or absence of carbohydrate side chains seems to be the only difference between these forms of proangiotensin. Both proteins consist of single polypeptide chains having apparent molecular weights of 52000 and 55000 and isoelectric points around 4.7 and 4.4, respectively. No significant difference between the proteins could be observed with respect to the amino-terminal amino acid sequence which was found to be the same (H2N-Asp-Arg-Val) as for angiotensin I and II. Furthermore, extensive digestion with renin, releasing the decapeptide angiotensin I, did not significantly reduce the molecular weights of both polypeptides. It can therefore be concluded that the angiotensin I peptide is located at the amino terminus of the prohormone. Kinetic constants measured for the release of angiotensin I by renin were found to be Km = 5.0 microM proangiotensin and V = 270 nmol of angiotensin I h-1 unit renin-1 for the concanavalin-A-binding form and Km = 5.6 microM proangiotensin and V = 250 nmol angiotensin I h-1 unit renin-1 for the prohormone which did not bind to concanavalin-A--Sepharose. The form of proangiotensin not bound to concanavalin-A--Sepharose was found to be more thermally labile (tm of 59.0 degrees C) than the form binding to concanavalin A (tm of 61.5 degrees C, where tm = temperature at which 50% reactivity is lost).
D000595:Amino Acid Sequence; D000808:Angiotensinogen / Q000097:blood* / Q000302:isolation & purification; D000809:Angiotensins / Q000097:blood*; D000818:Animals; D001798:Blood Proteins / Q000032:analysis; D002845:Chromatography / Q000379:methods; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D000809:Angiotensins; D001798:Blood Proteins; D000808:Angiotensinogen