Amino acid sequence of a fragment “histidine-rich peptide” released from bovine high molecular weight kininogen by plasma kallikrein and its biological activity

792

Conference Abstracts

Vol . 16, No .S

pancreas kallikrein 2-mercaptoethanol LMW-kininogen -----~ kinin-free ---------------~ polypeptide fragment protein (single chain, M.W . 7 .8 x 104 ) (M .W . 7 .6 x 104 ) (M .W . 7 .0 x 104 ) These results suggest that kinin is located on the loop of a polypeptide chain bridged by a disulfide bond in both kininogena, and that kinin is released from the central portion in HMW-kininogen, whereas kinin is released from near the terminal portion in LMW-kininogen . b) Cleavage with cyanogen bromide (CHBr ) : Bovine kininogena have a following kinin sequence ; -Met-Lys-BRADYKININ-Ser-Val-Gln-Val-Met- . Human kininogena were treated with CNBr and the product fractionatedon Sephadex G-75 . The results are summarized se follows : CNBr 1~fli-kininogen -- -~ kinin yielding peptide (M .W . 8 x 10 ) CNBr 2-mercaptoethanol LMW-kininogen ---a large fragment ------------~ kinin-yielding peptide (M .W . 4 .5 x 104) (M .W . 9 x 103) Human plasma kallikrein released rapidly Lys-bradykinin from the large fragment of LMW-kininogen . It seems that there ie no Met residue near the carboxyl terminal of the bradykinin sequence in two human kininogena . From these results, it seems that there are structural differences between HMW- sad LMW-human kininogens with respect to the locations of Met and Cya residues and the kinin sequence . AMINO ACID SEQUENCE OF A FRAGMENT ^HISTIDINE-RICH PEPTIDE" RELEASED FROM BOVINE HIGH MOLECLTIAR WEIGHT RININOGEN BY PLASMA KALLIRREIN AND ITS BIOLOGICAL ACTIVITY S . Iwanaga, M . Komiya, Y .N . Han, T . Suzukl, S . Oh-iehi, and M . Ratori Institute for Protein Research, Osaka University, Suits, Osaka-565 and Department of Pharmacology, School of Medicine, Ritasato University Sagamihara-228, Japan Two uncharacterized peptide fragmenta, which were released from bovine high molecular weight kininogen by plasma kallikrein, were isolated and their complete and partial amino acid sequences were determined . These fragmente con tained significantly high levels of hiatidine . One of the fragmente consisted of 41 residues and contained eleven each of hiatidine and glycine and seven of lysine . The amino acid sequence, determined by Edman degradation and standard enzymatic techniques, is : 5 1 10 15 H-Ser-Hie-Gly-Leu-Gly-ëis-Gly-His-Gln-Lys-Gln-His-Gly-Leu-Glq-Hie-Gly-His-Lys 20 25 30 35 His-Gly-Hie-Gly-Has-Gly-Lys-Hie-Lys-Asa-Lys-Gly-Lys-Ann-Asn-Gly-Lys-His-Tyr-Aep40 Trp-Arg-OH .

Vol . 16, No . 5

Conference Abstracts

79 3

The fragment had an extremely interesting feature in that the repeating These sequences, His-Gly-X or Gly-His-R, occur along the peptide chain . appeared 7 and 6 times, respectively, up to 26 residues from the N-terminal end . Moreover, the triple tetrapeptide sequences of Gly-His-Gly-His and double heptapeptide sequences of His-Gly-Leu-Gly-His-Gly-His were found in the N-terminal portion . On another fragment containing 10 histidine residues out of 67 amino acid residues, the partial N-terminal sequence was determined as follows : 67 1 5 11-Ser-Val -Gln-Val-Met-Lys-Thr-Glu-Gly-----ArgOH . Thin N-terminal sequence was the same as that of the C-terminal portion of the previously determined CNBr-fragment containing kallidin (Lys-Arg-Pro-Pro-GlyPhe-Ser-Fro-Phe-Arg- Ser-Val-Gln-Val-Met ) . The biological activity of the "histidine-rich peptide" is under investigation . It seems to have some inhibitory effect on the activation of preHageman factor . It is noteworthy that plasma kallikrein liberates histidine rich peptides from the kininogen, in addition to bradykinin . PROTEINS OF THE KININOGEN-RININ SYSTEM Ulla Homberg Department of Biochemistry, University of Hnlainki, Helsinki, Finland Molecular aspects on kininogen and urinary kallikrein are presented ; the function of alpha-2-macroglobulin as anti-kininogenase at high levels of endogenous plasminogen activation is blood is discussed. In recent studies kininogen (1) isolated from human pooled ACD plasma was found to by physico-chemically too closely related to alpha-2H5-glycoprotein to allow separation by conventional methods . Tables on the amino acid composition, specific activity and flow scheme on the heterogeneity of kininogen are presented . Immunologically pure kininogen was obtained with bioapecific immunosorbents prepared from antisera insolubilized with ethylchloroformate by removing alpha-2HS-glycoprotein, or by adsorption-desorption of kininogen, using the respective antibody-specific polymers . highly purified kininogen containing 30X or 14 .5X alpha-2H5 (2 .8X albumin) was purified comparing the two methods . The adsorption-desorption method was found to be favourable for immunization purposes in producing kininogen with high antigenic specificity . With less than 200 ug protein purified by immunosorption the kininogen precipitation titer was 1:8 after 7-8 weeks . With a freshly prepared polypar incubated with 800 u8 kininogen (14.5X alpha-2HS) 37-52X of the protein was desorbed with 2 M and 3 M sodium iodide in three consecutive adsorption-desorption procedures . Microparticles appearing with polymer used several times did not affect its antigenic capacity . Comparison of the disc gel patterns obtained showed several diffusely stained bands around the sharp main band, apparently of changed molecular forms of kininogen. This is suggested by the appearance with both materials of the similar patterns, which indicates that they are not products of nonspecific desorption from the antikininogen polymer . Kallikrein was isolated from rabbit urine, comparing the esterase enzymes and urokinase in urine from the bladder or after collection . Separation of urokinase was achieved by isoelectric focusing (2), also separating several esterase enzymes in collected urine . An albumin-associated esterase was identi-