Chiaki Moriwaki

Human Salivary Kallikrein and Liberation of Colostrokinin

Kallikreins from various sources have been studied by many investigators including Moriya et al. (1958; 1959a,b,c,d, e, f, g; 1963), to elucidate the physiological and pathological significance of the various kallikreins and of the vasodilator polypeptide, kallidin (or bradykinin), liberated by kallikreins.

Kallikrein Treatment of Male Infertility

Until recently, any approach for the treatment of male infertility had brought us little satisfactory results, if any. In 1973, Stuttgen showed that kallikrein increased the number of sperm in patients with oligospermia. Since then, many clinical studies have been performed successively with promising results (Schill, 1974). These findings have prompted a similar investigation in oligospermic patients and asthenospermic patients in our clinic to test the effectiveness of kallikrein on semen parameters. We have performed artificial insemination with husband’s semen for the treatment of male infertility, but conception rate has not been improved as we expected.

Purification of kallikrein from cat submaxillary gland

Cat submaxillary gland has been used in many investigations on kallikrein-kinin system. Though it has not yet been established, the physiological functions of kallikreins in various glands, such as the functional vasodilatation, have been studied mainly in cat submaxillary gland (1, 2, 3). Furthermore, the secretory cells or subcellular distribution has been studied in this gland (4). Meanwhile the isolation of submaxillary kallikrein has not yet been achieved. In a series of investigation on the proteases in the rat submaxillary gland, Ekfors, et al. obtained a kallikrein-like peptidase (5), but the identity of the enzyme with kallikrein was not clear because of the lack of study on the biological activity of the preparation. Fiedler, et al. described that hog submaxillary kallikrein was separated into a number of active components with isoelectric points in the range pH 3.3–4.4 (6), but the specific activity of their preparation was less than a half of that of purified hog pancreatic kallikrein. We attempted the isolation of kallikrein from the cat submaxillary gland and obtained a highly purified preparation which was homogeneous in disc electrophoresis.

Specificity of potato kallikrein inhibitors for kallikreins

Two polyvalent protease inhibitors, acting especially on plasma kallikrein, were isolated from potatoes. Some of their physicochemical and biological properties were investigated. Aus Kartoffeln wurden 2 polyvalente Proteaseninhibitoren im besonderen für Plasma-kallikrein isoliert. Einige physikochemische und biologische Eigenschaften der Inhibitoren wurden charakterisiert.

Effect of kallikrein-kinin system on ion transport across rat small intestine.

The flow of glucose and valine from the mucosal to the serosal side of rat intestine was stimulated significantly by kallikrein-kinin system. To elucidate the mechanism of this function the effects of kallidin on the transmural potential difference (PDt) between the serodal and the mucosal sides and the transepithelial resistance (Rt) of rat jejunum segment were investigated by electrophysiological procedures. The glucose-evoked PDt was not significantly altered by addition of kallidin to the mucosal fluid. Meanwhile, PDt which evoked by glucose was significantly shifted under the presence of 10-1-10(2) ng kallidin in the serosal fluid. The valine-evoked PDt was also changed by addition of 10 ng kallidin into the serosal fluid. However, the addition of glucose to the mucosal and kallidin to the serosal fluid did not affect on the Rt of the jejunum segment. These results suggest that kinin stimulated Na+ transport at the baso-lateral membrane of the intestinal absorptive cells, and the transports of other substances were enhanced consequently.

Some properties of acrosin, a kininogenase from sperm acrosome.

Acrosin is a protease in the mammalian sperm head, acrosome (Fritz et al., 1975) and its physiological role in the fertilization processes is thought to participate in the sperm penetration to ovum. This enzyme is classified as one of the serine protesses, such as trypsin and kallikreins (Fritz et ale, 1975). On the other hand, it has been reported that administrations of hog pancreatic kallikrein to male infertility patients, especially oligozoospermia and asthenozoospermia caused improvement of sperm counts and motility (Schill, 1977; Leidl, et al., 1974). Fritz et al. described that the kinin system enhances sperm motility, O2-consumption and fructolysis, in vitro (Schill, 1974).

Anti-bradykinin activity found in beet and some of its anti-inflammatory actions

[3] J.L. TURK, D.A. WILLOUGHBY and J.E. STEVENS, An Analysis of the Effects of some Types of AntiLymphocyte Sera on Contact Hypersensitivity and Certain Models of Inflammation, Immunology 14, 683-695 (1968). [4] R.J. PERPER, E.M. GLENN and R.E. MONOVlCrI, Separation of Anti-inflammatory and Immunosuppressive Activities in Heterologous Antilymphocyte Serum, Nature 223, 86-87 (1969). [5] H.L.F. CUR~Y and M. ZIFF, Suppression of Experimentally Induced-Polyarthritis in the Rat by Heterologous Antilymphocyte Serum, Lancet II, 889891 (1966). [6] M.E.J. BILLINGHAM, B.V. ROBINSON and J.M. GAUGAS, TWO Anti-inflammatory Components in Anti&mphocytic Serum, Nature 227, 276-277 (1977). [7] P.J. MORRIS and J.F. BUaKE, Antilymphocyte Serum and Staphylococcal Infection, Nature 214, 1138-1139 (1967). [8] J.E STEVENS and D.A. WmLOUGHBY, The Antiinflammatory Effect of Some Immunosuppressive Agents, J. Path. Bact. 97, 367-373 (1969). [9] J. GARCIA LEME, G.H. BECHARA and R. RIBEIRO DOS SANTOS, A Proinflammatory Factor in Lymphocytes. Its Role in the Development of Acute, Non-immunological Inflammatory Reactions, Br. J. exp. Path. 57, 377-386 (1976). [ 10] G.H. BECHARA, L. SUDO, R. RIBEIRO DOS SANTOS and J. GARCIA LEME, Modulation by Lymphocytes of the Vascular Effects Caused by Inflammatory Mediators and Carrageenin in the Rat, Br. J. exp. Path. 57, 497504 (1976). [11] R. RIBEIRO DOS SANTOS, J. GARCIA LEME, S.H. FERREIRA, G.H. BECHARA and L. SUDO, Does the Proinflammatory Factor in Lymphocytes (LpIF) Explain the Role of These Cells in Acute Inflammation? Agents and Actions 6, 690-693 (1976). [12] D.D. MCGREGOR and J.L. GOWANS, The Antibody Response of Rats Depleted of Lymphocytes by Chronic Drainage from the Thoracic Duct, J. exp. Med. 117, 303-320 (1963).

Mushroom kininase: Its application to the kallikrein-kinin system

The Japanese mushroom, Tricholoma conglobatum (Shimeji in Japanese), contains an extremely potent kininase. This kininase was purified 320-fold by ammonium sulfate fractionation and various chromatographic steps, the final preparation being homogeneous in disc electrophoresis. It splits the bradykinin molecule at the amino side of Phe(5) and of Phe(8) and the former bond was more easily cleaved than the latter [1]. The present paper deals with the stability of this kininase in the blood, its effect on plasma prekallikrein and its anti-inflammatory action. The kininase was incubated with rat plasma for 3 h at 30 ~ and this plasma-treated kininase was added to synthetic bradykinin together with 8-hydroxyquinoline which almost completely inhibits the kininase(s) in rat plasma but not the mushroom kininase. It was found that the kininase still held the activity to degrade bradykinin. Furthermore, 30 U of the mushroom kininase was injected i.v. into the rat and blood

Potent kininases obtained from mushrooms

PURIFICATION OF KALLIKREIN FROM CAT SUBMAXILLARY GLAND C. MORIWAKI, Y. HOJIMA, M. SCHACHTER Science University, of Tokyo, University of Alberta, Edmonton, Canada. The submaxillary gland is abundant in kallikrein and various investigations on the distribution and the function of kallikrein have been carried out on this gland of cat. However, its isolation has not been achieved yet, so we tried the purification of this kallikrein. From lyophilized powder (2600 mg) of a water extract from 33.5 g of the wet glands, the isolation of the kallikrein (12 mg) was accomplished by the following procedures: 50-75% acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-75 filtration and Ampholine isoelectric focusing. The final preparation gave the activities of 1260 KU, 24,5 and 11.6 pmoles BAEE and TAME hydrolysis per min per mg. The activities on other substrates (BTEE, BApNA and casein) were poor or negligible. The isoelectric points of the kallikrein were in range of pH 4.2-5.0, and this kallikrein seemed to be composed of several multiple forms with different pls. From these results, the properties of this kallikrein are simillar to those of the submaxillary glands of rat or porcine and other glandular kallikreins, but the migration of the kallikrein in disc electrophoresis was quite unique and it gave a broad and retarded one. This phenomenon may be ascribed to the carbohydrate in the kallikrein. Trasylol gave very weak inhibitory effect on this kallikrein as reported by Dr. Werles group. POTENT KININASES OBTAINED FROM MUSHROOMS H. MORIYA, K. KIZUKI, Y. HOJIMA and C MORIWAKI (Lab. of PhysioL Chem., Science Univ. of Tokyo, Shinjuku-ku Tokyo, Japan) Two kinds of potent kininases from mushrooms (ShimejiTricholoma conglobatum; and HaratakePsalliota campestris) were found and investigated. The contents of kininase activity in some mushrooms were examined. Mainly two kinds of enzymes were highly purified. Their properties, stability, actions against some synthetic substrates, behaviours for some inhibitors and so on were studied. The positions of peptide bond of bradykinin hydrolysis by these enzymes were examined by detection of DNS-modified products from bradykinin hydrolysed on TLC. One of enzymes showed the strongest activity of 2,284 kininase units per mg which value has never been known amoung those of kininases or proteases checked by the present method (#g bradykinin destroyed per min. at pH 7.4, 30~ These enzymes could be expected as useful tools for the study of kinin system in some experiments.

Potent kininases obtained from mushrooms.

In order to elucidate physiological functions or pathological meanings of the kallikrein-kinin system in the body, it might be an advantageous and important idea as one of the effective methods for approaching to find something strong and specific substance in the kinin system, for instance, to find specific kallikrein inhibitor, strong anti-kinin substance and potent kininase which would be sometimes developed to useful medicines for some diseases related to the kallikrein-kinin system.