Generation of kinin by plasma kallikrein and plasmin and the effect ofα(in1) antitrypsin and antithrombin III on the kininogenases

Abstract

A Synopsis of Kinin '75 -Abstracts low molecular weight Kgn I, which has the bradykinin (BK) sequence at its carboxyl terminus, and Kgn II, with the BK moiety inside the molecule (J.V. Pierce and M.E. Webster, in Hypotensive Peptides, E.G. Erd6s, N. Back, F. Sicuteri, eds., Springer, 1966), were equally good substrates for pepsin. All of the kinin was released in three minutes when 10 mg of Kgn B3.2c~ (containing ca. 15/Jg of BK/mg, as determined with trypsin) was incubated with 50/Jg of 2X crystallized pepsin at pH 2.0 and 37 ~ . The released kinin, equivalent to 18 #g of BK on the isolated guinea pig ileum, was stable in the incubation mixture. It was inactivated by carboxypeptidase B and could be converted to an 8-fold more active peptide upon incubation with human plasma aminopeptidase or dipeptidyl aminopeptidase I (DAP-I). The peptic Kinin was separated from the bulk of protein by gel filtration on Sephadex G-25 and was further purified by SP-Sephadex G-25 and CM-cellulose chromatography. The protein fraction did not generate any additional kinin upon incubation with trypsin or human plasma kallikrein. Several criteria were used to identify the purified peptic kinin as MLBK: a) it has the same retention volume as MLBK on an analytical SP-Sephadex C-25 column which is able to resolve BK, LBK, and MLBK; b) it was converted by treating with DAP-I to BK, identified by the 8-fold activity increase and by its now having the same retention volume as BK on the SP-Sephadex C-25 columnn; and c) it had the same amino acid composition as MLBK. The kinin released from other pure Kgn's by pepsin was also identified as MLBK by some of the above criteria. Pepsin, like trypsin and plasmin, cleaves several peptide bonds in Kgn's other than the bonds involved in the release of MLBK. The kinin-releasing activity of pepsin extends to pH 5.0 and is inhibited by pepstatin at both pH 2.0 and 5.0 Our highly purified preparations retained full Kgn activity at pH 2.0 after several days at room temperature (ca. 25~ MLBK was first isolated from bovine plasma which had been dialyzed against 0.01 M HCI and then incubated at pH 7.5 (D.F. Elliott and G.P. Lewis, Biochem. J. 95, 435, 1965). Since plasma contains pepsinogen, its activation in the acid dialysis step could explain the release of MBLK observed by Elliott and Lewis. GENERATION OF KININ BY PLASMA KALLIKREIN AND PLASMIN AND THE EFFECT OF oq ANTITRYPSIN AND ANTITHROMBIN III ON THE KININOGENASES M. FLAVIO, HABAL, E. CLEMENT, BURROWES and Z HENRY, MOVAT Division of Experimental Pathology, Department of Pathology, University of Toronto, Toronto, CanadaPlasma prekallikrein and plasminogen were purified by anion and cation exchange chromatography, gel filtration and affinity chromatography. The zymogens were activated with highly purified prekallikrein activator and streptokinase respectively. Low (LMW) and high (HMW) molecular weight kininogens purified by a series of chromatographic steps as described before (Biochem. Pharmacol. 23: 2291, 1974). Alpha1 -antitrypsin and antithrombin