Hisao Kato

Bradykinin-potentiating peptides from the venom ofAgkistrodon halys blomhqffii

Aus dem Gift vonAgkistrodon halys blomhoffii konnten durch Säulenchromatographie an Sephadex G-100, G-25 und CM-Sephadex C-50 fünf Peptidkomponenten isoliert werden, welche die kontrahierende Wirkung von Bradykinin auf den isolierten Meerschweinchendarm signifikant erhöhen.

The isolation and amino acid sequences of new pyroglutamylpeptides from snake venoms.

Die Strukturaufklärung von zwei neuen tryptophanhaltigen Peptiden im Schlangengift vonAgkistrodon halys blomhoffii ergeben:l-Pyroglutamyl-l-Glutaminyl-l-Tryptophan undl-Pyroglutamyl-l-Asparaginyl-l-Tryptophan. Diese Peptide sind in Schlangengiften der Viperidae- und Crotalidae-Arten verbreitet.

Fluorogenic Peptide Substrates for Proteases in Blood Coagulation, Kallikrein-Kinin and Fibrinolysis Systems

Mammalian plasmas contain a number of “serine-active site” trypsin-like proteinases, which participate in blood coagulation, kallikrein-kinin, fibrinolysis and complement systems. The proteinases have an ability to cleave selectively proteins, that is, limited proteolysis, and catalyze the cascade reactions, which involve several sequential transformations of proenzymes to enzymes (Davie et al. 1969). Table i shows the amino acid sequences around the scissile bonds of natural substrates attacked by these proteinases. It has been well known that the amino acid residues preceding the scissile bond, P2 and P3 sites, in the substrates, are of importance for the enzyme-substrate interaction (Blomback, 1970). For instance, a part of the sequence, Asp-Asp-Asp-Lys, which is located in the NH2-terminal portion of trypsinogen, has been known to comprise the substrate recognition sites and specificity sequence for enterokinase (Ottesen, 1967). Similarly, Factor Xa is presumed to recognize the tetrapeptide sequence Ile-Glu-Gly-Arg located close to the cleavage site required for the activation of bovine prothrombin (Magnusson et al. 1975). Based on this idea, several peptidyl-p-nitroanilides, so called “chromogenic substrate”, which suit a specificity requirement of proteinases,

Structure of bradykinin-potentiating peptide containing tryptophan from the venom ofAgkistrodon halys blomhoffii

Es wird über die Strukturaufklärung eines Bradykinin-potenzierenden Peptids aus dem Gift vonAgkistrodon halys blomhoffii berichtet.

Purification of guinea-pig plasma prekallikrein. Activation by prekallikrein activator derived from guniea-pig skin

Prekallikrein was purified from guinea-pig plasma. The prekallikrein appeared homogeneous as a single-chain protein on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. The apparent molecular weight was 82 000 by SDS-polyacrylamide gel electrophoresis, 99 000 by gel filtration on a Sephadex G-150 column and 84 500 (protein part) by amino acid analysis. The isoelectric point was approx. 9.0. The purification method yielded 3.8 mg (A280 3.800) of prekallikrein from 500 ml of plasma. Kallikrein was generated from the prekallikrein by limited proteolytic action of a prekallikrein activator which was derived from guinea-pig skin. From analysis using SDS-polyacrylamide gel electrophoresis, the kallikrein has two fragments with apparent molecular weights of 52 000 and 40 000 which are linked by disulfide bond(s). The 40 000 molecular weight fragment was shown to incorporate [3H]diisopropylfluorophosphate. The kallikrein hydrolyzed the synthetic substrates containing the Phe-Arg sequence at the COOH-terminal, and it cleaved carbobenzyloxy-Phe-Arg-4-methylcoumaryl-7-amide more readily than Pro-Phe-Arg-methylcoumaryl-7-amide. The Km for the kallikrein with carbobenzyloxy-Phe-Arg-methylcoumaryl amide was 2 times 104 M. Also, the kallikrein showed negligible activities on peptide-methylcoumaryl amide-substrate for alpha-thrombin, Factor Xa or plasmin.

Purification and properties of rats stomach kallikrein

Kallikrein (EC 3.4.21.8) was purified from rat stomach by column chromatography on p-aminobenzamidine-Sepharose, DEAE-Sephadex A-50 and Sephadex G-150 and by isoelectric focusing, measuring its activities to hydrolyse l-prolyl-l-phenylalanyl-l-arginine-4-methyl-coumaryl-7-amide and to release kinin from heat-treated rat plasma. The purified stomach kallikrein showed a single band on polyacrylamide gel electrophoresis at pH 7.0. n nIts molecular weight was calculated to be 29 000 by gel-filtration on a column of Sephadex G-50. The kallikrein was stable between pH 6–11 and hydrolyzed l-prolyl-l-phenylalanyl-l-arginine-4-methyl-coumaryl-7-amide optimally at pH 11.0. The l-prolyl-l-phenylalanyl-l-arginine-4-methyl-coumaryl-7-amide hydrolyzing activity of rat stomach kallikrein was inhibited by diisopropyl fluorophosphate and Trasylol, but not by trypsin inhibitors from soybean, lima bean and ovomucoid. These properties of rat stomach kallikrein are different from those of partially purified rat plasma kallikrein, but similar to those of grandular kallikreins from other species. n nFrom these results, it was concluded that kallikrein is present in rat stomach and that it can be classified as a glandular kallikrein.

Identification of Ser-Leu-Met-Lys-bradykinin isolated from chemically modified high-molecular-weight bovine kininogen

Bovine blood plasma contains at least two kininogens, viz., high-molecular weight (HMW) and low-molecular weight (LMW) kininogen; each consists of a single polypeptide chain with a molecular weight of 76 000 or 50 000, respectively [I] . Both of these include the bradykinin moiety in their inner portions surrounded by a disulfide loop [2]. Upon incubation with plasma kallikrein or snake venom kininogenase, they yield the corresponding kinin-free proteins which consist of a heavy and a light chain [2] derived, respectively, from the NHzand COOH-terminal portions of the parent molecule [3] . The heavy chains have the COOH-terminal sequence -Leu-MetLysOH [4], giving rise to the speculation that the heavy chain is contiguous with the kinin moiety in both kininogens, inasmuch as Met-Lys-bradykinin has been isolated from bovine plasma [5]. However, this sequence does not agree with those of the two kinin-containing peptides, Ser-Arg-Met-Lysbradykinin and Gly-Arg-Met-Lys-bradykinin, which were isolated by Hochstrasser and Werle [6] from the peptic digest of bovine plasma Cohn fraction IV-6. To resolve this contradiction, we have re-examined bovine kininogen and established the sequence of residues preceding the kinin moiety. The strategy used was to prepare a chemically modified HMW kininogen, digest it with pancreatic kallikrein, isolate the kinincontaining peptide from the digest, and determine its amino acid sequence. The results indicate that the sequence is Ser-Lcu-Met-Lys-bradykinin, showing that the COOH-terminus of the heavy chain is indeed juxtaposed to the kinin moiety. 2. Materials and methods

Primary structure of bovine high molecular weight kininogen: Chemical compositions of kinin-free kininogen and peptide fragments released by plasma kallikrein

High molecular weight (HMW) kininogen found in mammalian blood plasma is one of the functional glycoproteins, which participates in the kallikreinkinin system [l] . Quite recently, a new function of this protein has been suggested by findings of Flaujeac and Williams traits with a deficiency of the kininogen [2,3] . The deficient plasma does not release kinin upon incubation of kallikrein and also has a prolonged activated partial thromboplastin time and inability to form plasmin. These suggest a participation of the kininogen on the intrinsic blood coagulation and fibrinolysis, in addition to the kinin-forming system. In order to realize these physiological functions, it seems important to study the chemical structure of kininogen. Previously, we reported that a few of peptide fragments, in addition to kinin, were liberated from HMW kininogen on the digestion with plasma kallikrein [4]. One of the fragments was found to contain an abnormally high level of histidine (named tentatively histidine-rich peptide), and its amino acid sequence was established [5] . This communication describes further studies on the chemical compositions of kinin-free kininogen and peptide fragments produced from HMW kininogen by plasma kallikrein.

Bovine plasma HMW and LMW kininogens: isolation and characterization of the polypeptide fragments produced by plasma and tissue kallikreins.

There exist at least two types of kininogens with different molecular weight in mammalian blood plasmas, which are named high molecular weight (HMW) and low molecular weight (LMW) kininogens (1). Although their functions in kallikrein-kinin system remain to be investigated, it has been speculated that HMW kininogen participates in the intrinsic kinin releasing system and Imw kininogen in the extrinsic kinin-releasing system. Quite recently, a new function of kininogen has been suggested by findings of Flaujeac (2), Williams (3), and Fitzgerald (4) traits with a deficiency of kininogen. These plasmas do not release kinin appreciably upon incubation of plasma kallikrein and also has a prolonged activated partial thromboplastin time and inability to form plasmin. These facts suggest the participation of the kininogen in the intrinsic blood coagulation and fibrinolysis, in addition to the kinin-forming system.

Estimation of Urinary Kininogenase Activity Using Bovine Serum Low Molecular Weight Kininogen

Estimation of urinary kininogenase activity by radioimmunoassay of generated kinin was studied. Bovine serum low molecular weight kininogen was proved not to cross-react with kallidin antibody and also bradykinin antibody. This kininogen was used as substrate measuring urinary kininogenase activity. Separation of released kinin from the kininogen was not required in the present method. Urinary kallikrein activity was found to be significantly decreased in essential hypertension, in chronic glomerulonephritis and in patients who had received renal transplantation. On the contrary, an increase in urinary kallikrein was found in primary aldosteronism and in Bartters syndrome. The present method was very useful for measuring kininogenase activity.