Kininase Activity Research Articles

Mechanism of action of cyclokinins

Cyclic analogs of bradykinin (CBK) and kallidin (CK) have a weak myotropic activity and a marked and prolonged hypotensive effect unlike linear bradykinin (BK) and kallidin (K) which produce a short-term hypotension and considerable contraction of rat uterus smooth muscles. Myotropic effects of BK and CK were significantly inhibited by phentolamine, methysergide, papaverine and verapamil. Atropine, diphenhydramine and propranolol have no influence on the kinin-induced myotropic responses. The prolonged decrease in blood pressure induced by CBK and CK is observed in un- and anesthetized normotensive, spontaneously hypertensive rats and rats with renovascular hypertension and is absent from anesthetized cats and guinea-pigs. This indicates the species specificity of cyclokinins. Indomethacin, diphenhydramine and methysergide failed to influence the BK- and CK-induced hypotensive effects on anesthetized rats. CaCl2 did not influence the magnitude of the hypotensive effect of BK and CK, however, it significantly shortened the duration of the CK-induced hypotensive effect. In vitro CBK and CK inhibited activity of kininase II in a similar manner (at a concentration range of 10(-5) M) but to a less extent than BK (10(-7)-10(-6) M). It is suggested that the hypotensive effect of CK is mediated at least partly via Ca2+-dependent systems and inhibition of kininase II.

A kininase and a kinin-converting enzyme: two distinct alpha aminoacyl peptide hydrolases from bovine lung.

Two closely related but different aminopeptidases from bovine lung have been isolated and characterized. The first aminopeptidase, which removes the N-terminal arginine residue from L-arginyl-L-prolyl-L-proline, bradykinin, and des-[Arg9]-bradykinin, has kininase activity; it has a pH optimum of 8.0, is stimulated by Mn2+, and its molecular weight in dilute buffers is slightly greater than 240,000 daltons. The second aminopeptidase, which converts kallidin to bradykinin, has kinin-converting activity; it has a pH optimum of 6.8, is stimulated by Co2+, and its molecular weight in dilute buffers is 250,000 daltons. The kinin-converting enzyme is blocked from action when the N-terminal penultimate residue is proline.

Fluorometric assay of kininase activities in rat tissues

A simple method for the assay of bradykinin (BK)-degrading enzymes was investigated. The procedure of the method includes enzymatic degradation of BK, separation of the residual BK on a small P-cellulose column (0.6 × 3 cm), and its fluorometrical determination based on the reaction with fluorescamine. BK was separated completely from its fragments produced during enzymatic reaction by the column chromatography. The recovery rate of BK was 96 ± 3%. Quantitative determinations could be carried out on 0.2 nmol of BK, at least in the fluorometry. This method was available for the assay of the enzymes in tissue homogenates as well as in purified preparations, and its usefulness for the study of the enzymes is presented.

Effect of dihydralazine on the renal kallikrein-kinin system of the rat

Dihydralazine (0.1 mg/kg), injected intravenously into male Sprague-Dawley rats, caused a decrease in mean arterial blood pressure and an increase in renal plasma flow, while urine volume remained unchanged. Dihydralazine had no effect on kallikrein excretion in the urine and on kallikrein activity in the renal cortex. No correlation was found between renal kallikrein and either renal plasma flow or mean arterial blood pressure. The excretion of kinins in the urine rose markedly after the administration of dihydralazine; no correlation between urinary kinins and urinary or renal kallikrein was observed. Dihydralazine had no influence on the kininogen content of blood-free renal cortex. The enzymatic activity of kininase II in renal cortex was not impaired by dihydralazine. It is suggested that the increased formation of kinins within the kidney could be involved in the vasodilating and blood pressure lowering effect of dihydralazine.

A highly sensitive fluorometric assay of bradykinin and its fragments released by a kininase

The activity of kininase was studied using both techniques of fluorometry and thin-layer chromatography. The enzyme purified from rat brain, cleaved the bradykinin (BK) molecule at the peptide bond Phe 5-Ser 6 releasing two peptide fragments Arg-Pro-Pro-Gly-Phe and Ser-Pro-Phe-Arg. Conversion of these peptides and BK to fluorescent compounds using 1-dimethylaminonaphthalene-5-sulfonyl (DNS)-chloride, made it possible to detect the peptides on a silica gel plate at 0.25 – 2.0 nmol. These dansylated peptides were clearly separated using a mixture of toluene:monochloroethanol:25% NH 3 (6:10:0.5, v v% ) as developing solvent. With the aid of this solvent and dansylation, it was possible to determine the rate of the enzymatic reaction by the appearance of products and/or the disappearance of the substrate on the chromatogram. The kinetics of the enzyme were examined using this method.

Inhibition of rabbit tissue kininase by anti-(endo-oligopeptidase A) antibodies.

Highly purified rabbit brain endo-oligopeptidase A injected into goats produced, after 60 days of immunization, antisera that specifically inhibit purified rabbit brain endo-oligopeptidase A. An immunoreactive kininase having the same specificity as rabbit brain endo-oligopeptidase A for bradykinin was detected in several rabbit tissues. The highest amount of this immunoreactive kininase was found in the 25000 g supernatant fraction (S fraction) of heart, liver, skeletal muscle, ovary, brain and testis homogenates, corresponding to 89, 86, 78, 59, 56 and 53% respectively of the whole kininase activity found in the S fraction.

Regional distribution of kininase in rat brain.

Kininase activity, which inactivates kinins, was measured in seven regions of the rat brain (i.e., the cerebral cortex, cerebellum, striatum, midbrain, hippocampus, hypothalamus, medulla oblongata), and in the spinal cord with a bioassay method using bradykinin as the substrate. Specific kininase activities in the cerebellum and striatum were higher than those in the other five regions or the spinal cord. Angiotensin-converting enzyme activity, which was measured fluorometrically using Hip-His-Leu as substrate, showed high activity in the striatum and cerebellum. These findings suggest that the presence of high concentrations of peptidases plays a role in the degradation of kinins and/or other peptides in these areas.

Effect of stretching on the sensitivity of the guinea pig ileum to bradykinin and on its modification by bradykinin potentiating peptides

The sensitivity of the guinea pig isolated ileum to bradykinin, but not to other agonists, was increased ca. 2-fold during the 3–4 h following mounting of the preparation under 1 g load. Concomitantly, a decrease was observed in the bradykinin potentiating effect of BPP9a, and of potentiator B but not potentiator C. This decrease was observed only with analogues of BPP9a or potentiator B which retained one or both of the basic amino acid residues in these peptides. Similar stretching of the rat isolated uretus did not affect bradykinin sensitivity or the potency of bradykinin potentiating peptides. Kininase activity of the ileum significantly increased during the 4 h period after mounting of the loaded preparation, but was not affected by treatment with BPP9a. It is proposed that bradykinin sensitivity is favoured by the changes in sodium and potassium transport in the cell membrane caused by stretching of the ileum, and that a similar mechanism may be partly responsible for the action of bradykinin potentiating peptides.

Kininases of human polymorphonuclear leucocytes

Four kininases were isolated from human polymorphonuclear (PMN) leucocytes by gel filitration. The molecular weights of kininases I, II, III and IV were 270,000, 90,000, 47,000 and 4000, respectively. No appreciable difference among these kininases was observed in the optimum pH between 7.5 and 8.5. The K m value of the four kininases for bradykinin hydrolysis was 0.20 × 10 −6 M. These kininases did not hydrolyse hippuryl-hystidyl-leucine and hippuryl- l-lysine, in sharp contrast to plasma kininase. The kininase activity of the enzymes was completely destroyed by neutral proteases in the PMN cell lysosomal granule extract or after heating at 56° for 30 min. Kininases II, III and IV were released extracellularly during phagocytosis of kaolin pellets (200 μg/ml). The properties of the PMN cell kininases are discussed in relation to their possible role in the kinin system at the imflammatory site.

Angiotensin I converting enzyme (kininase II) of the brush border of human and swine intestine

Mucosal brush border of human and swine small intestine is rich in angiotensin I converting enzyme or kininase II (ACE). The brush border of the intestinal mucosa was purified by centrifugation over a discontinuous glycerol gradient. Transmission electron micrographs showed that 90 per cent of the isolated vesicles had a trilaminar membrane structure and glycocalyx, characteristic of intestinal brush border. No significant contamination by other subcellular particles was evident. In the final purified preparation, the brush border marker enzymes sucrase, trehalase and alkaline phosphatase were enriched 23-, 18- and 17-fold from human intestine and 27-, 26- and 20-fold from swine tissue. ACE was highly concentrated in the human and swine brush border. The specific activity of ACE in the human and swine brush border fractions was enriched 17- and 7.6-fold over the crude homogenate. Kininase activity was demonstrated by bioassay. Captopril, the orally active specific inhibitor of ACE, inhibited the enzyme; its I 50 was 3 × 10 −9. Antibody to swine kidney ACE cross-reacted with swine intestinal enzyme as shown in rocket immunoelectrophoresis, indicating that the enzymes from kidney and from intestine have common antigenic determinants and that the enzyme is concentrated on the brush border membrane. Because of the abundant presence of ACE in the intestine, interference in the functions of this enzyme may occur with chronic captopril therapy.

Kallikrein-kinin system and adenosine metabolism system of blood and heart and their changes at hypothalamic-hypophyseal stimulation in rabbits of different age.

It was shown that kallikreinogen content was decreased in blood of old rabbits, kallikrein activity was somewhat increased, kininase activity was significantly decreased. In old animals, adenosine metabolism was activated, this being evident from the rise of 5'-nucleotidase and adenosine deaminase activity in blood and myocardium. Hypothalamic stimulation resulted in significant activation of the kallikrein-kinin system and sharp increase of kallikrein activity, the shifts being less marked in old animals than in adults. In adult animals, vasopressin administration elicited more marked activation of the kallikrein-kinin and adenosine metabolism systems.

Studies on the components of kallikrein-kinin system and treatment of male infertility.

The kininase activity in the seminal plasma was identified with kininase II (angiotensin-converting enzyme) by thin layer chromatography and its activity of infertile patients was significantly lower than in normozoospermic patients. The amount of proteinase inhibitor in the seminal plasma was 7.0 ± 0.9 trypsin inhibitor unit/ml, however no correlation was found between its activity and semen quality.The effects of bradykinin and kallikrein on the velocity and motility of washed human spermatozoa were measured by multiple exposure photography method. The addition bradykinin at each concentration of 0.1ng/ml and 1.0ng/ml to the washed spermatozoa resulted in an increase of velocity and motility. The same effect as bradykinin on the spermatozoa was found in the presence of 0.1 KU/ml and 1.0 KU/ml of kallikrein.In order to determine the effect of kallikrein on the concentrations of seminal fluid components, infertile men were treated orally for 6 months. Following determinations of volume, motility and sperm density, the concen-trations of kininase II, proteinase inhibitor, lactate dehydrogenase and acid phosphatase were measured. Sperm motility and density were increased dur-ing treatment. Mean values of the parameters in the ejaculate unchanged, with the exception of kininase II, which exhibited significantly higher con-centration under treatment.Of a total of 131 men treated, 23 pregnancies (17.6%) is established be-tween the first and the sixth month.Thus, present work shows that the components of kallikrein-kinin system participate in spermatogenesis and sperm motility.

Kallikrein and kininase activities in whole saliva from patients with periodontal disease (author's transl)

The kinins, a group of biologically active polypeptides, are generally considered as important chemical mediators in the inflammatory process. However, the role of these mediators in periodontal disease still remains unknown.To make these points clear, the present study was performed to measure kallikrein and bradykininase activities in whole saliva from patients with periodontal desease, and to correlate these data with various clinical conditions such as GI-score, OHI-score, Pocket depth and Bone loss score.The kallikrein activity in the samples was measured by colorimetric method of Moriya et al. and fluorometric method of Matsuda et al., Both methods were compared to find out the better way for the mesurement of kallikrein activity.The bradykininase activity in the samples was assayed by the biological method of Takeya et al., The enzyme activity was expressed as the percentage of inactivation of bradykinin.The following results were obtained:1. Kallikrein was measured in the range from 10 to 40μg/ml by colorimetric method, however, much lower amount of kallikrein, 0.5 to 12μg/ml, could be detected by fluorometric method.2. Statistically significant positive correlations were found between kallikrein activity and all the clinical conditions examined (p<0.001).3. Statistically significant negative correlations were obtaind between bradykininase activity and all the clinical conditions examined (-p<0.001), and also between kallikrein activity and bradykininase activity (-p<0.001).4. The kallikrein activities in the saliva increased following the progress of periodontal disease, but the bradykininase activities remarkably decreased.

Screening for rabbit brain neuropeptide-metabolizing peptidases. Inhibition of endopeptidase B by bradykinin potentiating peptide 9a (SQ 20881).

Neutral thiol-activated peptidases present in the pH 5-soluble fraction of rabbit brain (separated by step-elution chromatography on diethylaminoethyl cellulose) were screened for the hydrolysis of bradykinin. Lys-bradykinin, Met-Lys-bradykinin, angiotensin I, angiotensin II, substance P, luteinizing hormone-releasing hormone (LH-RH), and neurotensin by bioassay. The column effluent was monitored for bradykinin inactivation and arylamidase activity and combined in six pools on the basis of bradykinin inactivation. The pools were characterized by determining the peptide fragments and amino acids released from bradykinin with an amino acid analyzer. Pools 1 through 3 contained 80% of the kininase activity and essentially all of the endopeptidase A and B activity, whereas pools 4 through 6 accounted for 98% of the recovered arylamidase activity. Bradykinin, angiotensin I, angiotensin II, and substance P were inactivated by all the pools, whereas LH-RH and neurotensin were inactivated by pools 3 and 4, and pools 3, 4, and 5, respectively. These data show that rabbit brain contains peptidases having some selectivity for the inactivation of neuropeptides. Endopeptidase B purified from pool 3 is inhibited by bradykinin-potentiating peptide 9a (BPP9a, SQ 20881) (< Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro), a competitive inhibitor of the hydrolysis of bradykinin (Km = 3.5 X 10(-5) M, Ki = 3 X 10(-6) M) which also completely inhibits the inactivation of LH-RH.

An experimental study on the effects of the chronic administration of an angiotensin converting enzyme inhibitor (captopril) on blood pressure and the renin-angiotensin-aldosterone system (author's transl)

The chronic effects of the oral administration of an angiotensin converting enzyme inhibitor (captopril, 63-80 mg/kg/day) on blood pressure and the renin-angiotensin-aldosterone system were studied in normotensive male Wistar rats. The blood pressure (BP), plasma renin activity (PRA), plasma renin concentration (PRC), plasma renin substrate concentration (PRS), plasma aldosterone concentration (PAC), and renal renin content (RRC) were measured 2, 9, 19, 29 and 58 days after the administration of the agent. Blood pressure was determined in unanesthetized rats, and blood samples were obtained by decapitation. Furthermore, blood pressure responses to bolus injections of angiotensin II (AII, 80 ng/kg), angiotensin I (AI, 80 ng/kg) and bradykinin (BK, 10 micrograms/kg) were examined in unanesthetized rats which had been given the agent for 20 days. The BP of the rats which had been given captopril for 9 days or more was significantly lower than in the control rats. The reduction in heart weight in the captopril rats reached a statistically significant level 58 days after the administration of the agent. The PRA and the PRC markedly increased, and the PRS and the PAC both decreased in the captopril rats. The RRC which was reduced after 2 days of captopril administration markedly increased thereafter. In the captopril rats, significant negative correlations were observed between PRC and PRS (r=-0.43, p<0.01), and between BP and PRC (r=-0.60, p<0.001). The PRC significantly correlated with the RRC in the control rats (r=-0.44, p<0.01) while such a relationship did not exist in the captopril rats. Although the pressor responses to AII were similar in the captopril and the control rats, the responses to AI were reduced to 50% of the responses to AII in the captopril rats. The blood pressure reduction in response to BK in the captopril rats was 2.3 times as great as that in the control rats. Thus, it is suggested that the increases in PRA, PRC and RRC in the captopril rats may be related to both the blood pressure reduction and interruption of the negative feedback inhibition of renin synthesis and release by AII, and that the decrease in PRS in the captopril rats is due to the increased consumption and probably to the decreased rate of substrate production in the liver which is secondary to the decrease in plasma AII. The lack of significant positive correlation between PRC and RRC in the captopril rats seems to demonstrate that captopril may modify the relationship between them. The results also show that the chronic administration of captopril lowers blood pressure in normotensive animals. The blood pressure reduction by captopril may be related to the decreased kininase activity which is suggested by the enhanced depressor responses to BK, as well as the lowered plasma level of AII.

A study of kininase activity in pig liver [proceedings

A study of kininase activity in pig liver [proceedings

High concentrations of free kinins and kinin system components in abdominal transudate of a patient with nephrotic syndrome

High levels of bradykinin (60–80 ng/ml) were found in abdominal transudate from a patient with nephrotic syndrome caused by chronic glomerulonephritis. The abdominal transudate contained neutral kininogenase and its precursor, identified with plasma kallikrein and prekallikrein, respectively, as well as both forms of kininogen, the low-molecular-weight form predominating. The abdominal transudate was characterized also by very low kininase activity and low levels of α 1-antitrypsin (0.46 g/1) and α 2-macroglobulin. Large amounts of very low density lipoproteins were present in the transudate. Despite the difference in total protein content between the abdominal transudate and the patient's serum (4.3 g/1 and 48 g/1, respectively) their protein fraction composition was similar. The data obtained suggest that bradykinin is important in maintenance of long-lasting blood vessel hyperpermeability, which, in turn, is a driving force in the pathogenesis of refractory nephrotic edema.

Assay methods for prekallikrein and kininogens and their applications.

Prekallikrein activity in plasma was assayed using a synthetic peptidyl fluorogenic substrate (carbobenzoxy-L-phenylalanyl-L-arginine 4-methylcoumarinyl-7-amide), after activation of prekallikrein by acetone and kaolin. For total kininogen assay, the pretreatment of plasma at pH 2.0 was the best to eliminate bradykinin potentiators and kininase activity, before addition of trypsin to convert kininogen to bradykinin. Assay method of high molecular weight (HMW) kininogen was established by conversion of HMW-kininogen to bradykinin through activation of Hageman factor by glass powder and that of low molecular weight (LMW) kininogen was also by treatment of HMW-kininogen-depleted plasma in the same way as that for total kininogen. The marked reduction of prekallikrein and HMW-kininogen, not of LMW-kininogen, was found in pleural fluid of rat carrageenin pleurisy, and in plasma after i.v. injection of bromelain in rats. Members of the pedigree of hereditary angioneurotic edema patients also show low levels of prekallikrein and kininogens in plasma.

Kinin inactivating enzyme from mushroom Tricholoma conglobatum. II. Some enzymatic properties of the purified enzyme.

Some enzymatic properties of the new potent kinin inactivating enzyme (named as Shimeji kininase) which was purified from a mushroom Tricholoma conglobatum (Shimeji, in Japanese) were studied. This enzyme inactivated bradykinin, kallidin and Met-Lys-bradykinin by the ratio of 1.0 : 1.0 : 1.5, respectively, calculated as the molar ratio, and its Km value for bradykinin hydrolysis was 1.9 μM. Isoelectric point and molecular weight of the enzyme were 4.5 and 6.6×104, respectively. Optimum pH of this enzyme was 7.4 and it was comparatively stable at pH 6-9. This enzyme was thermolabile, i.e., more than 50% of the activity vanished by heating at 45° for 10 min and completely vanished at 60° for 10 min. The chelating agents, trasylol, SBTI and bradykinin potentiator B had no detectable influence upon the kininase activity of the enzyme. TLCK, TPCK and DFP weakly inhibited the enzymatic activity. The mercurials strongly inhibited the enzymatic activity of it and the inhibited activity was restored by adding excess amount of 2-mercaptoethanol. Accordingly, sulfhydryl groups in the enzyme molecule are requisite for its kininase activity and this enzyme may be classified as the thiol enzyme. The enzyme hydrolyzed the amino terminal substituted peptides such as Bz- and Z-derivatives while N-terminal free di- or tri-peptides were hardly hydrolyzed by it.

口腔清掃停止による全唾液, 歯垢および歯肉溝浸出液中の Kinins および Kininase の推移について

The present study was performed to elucidate the variation of kinin contents and kininase activities in the saliva, plaque and gingival fluid of the healthy people without oral hygiene.Nine male dental students, aged 20-24, were the experimental subjects. All of the participants had a tooth scaling and polishing, and then they were instructed not to brush their teeth for the following 10 days. At the 11th day, they were allowed to have a tooth brushing (Rolling method) 3 times a days for 5 days. The collection of samples (saliva, plaque and gingival fluid) and clinical examinations were performed daily for the entire experimental period (16 days). Kinin contents and bradykininase activities in the samples were assayed by a biological method of Takeya et al. The bradykininase activity was determined by measuring the percent of inactivation of added bradykinin (1μg/ml).The following results were obtained;1. The plaque index, gingival index and amount of gingival fluid were increased throughtout the period without oral hygiene (10 days). These clinical scores showed a rapid decline when the oral hygiene procedure was resumed.2. Kinin contents in the samples were almost zero for 5 or 6 days after stopping of the oral hygiene, and then it increased gradually for the following 4 or 5 days. The oral hygiene procedure was resumed at 10 days, and after that kinin contents decreased rapidly until these were not measured in the samples.3. Bradykininase activities in the samples decreased gradually after the oral hygiene was stopped, and these enzyme activities disappeared at 4-5 days after stopping of the oral hygiene. But, after the resume of oral hygiene, the enzyme activities rapidly increased to the level of the first days of the experiment.