Kininogenase Activity Research Articles

Mechanisms of suppression of renal kallikrein activity in low renin essential hypertension and renoparenchymal hypertension.

The mechanism of suppression of renal kallikrein activity in low renin essential hypertensive and renoparenchymal hypertensive patients was investigated in this study. From Sephadex G-200 column chromatography studies, a single kallikrein peak was observed in both kallikrein radioimmunoassay and kininogenase activity in all samples from normal subjects, low renin essential hypertensive and renoparenchymal hypertensive patients, and in purified kallikrein solution. The enzyme-specific activity around the kallikrein peak in all urine samples from each group was significantly lower than that in purified kallikrein, and a significantly lower specific activity was found in both patient groups than was found in normal subjects. Moreover, it was also recognized that the specific activity of kallikrein decreased in all cases with the increase of the molecular weight of kallikrein, and this tendency was observed more obviously in the low renin essential hypertensive and renoparenchymal hypertensive patients than in the normal subjects. These results suggest the presence of a kallikrein-specific inhibitor with a low molecular weight in human urine, although the possibility of a variant form of kallikrein cannot be excluded.

Identification of a tissue kallikrein in human polymorphonuclear leucocytes.

We have identified a tissue kallikrein in polymorphonuclear (PMN) leucocytes of normal human blood and bone marrow by immunocytochemistry, radioimmunoassay and enzymology. Immunoreactive tissue kallikrein was visualized in the mature neutrophil leucocytes and in immature forms such as metamyelocytes and myelocytes. No tissue kallikrein was detected in eosinophil leucocytes, lymphocytes, macrophages, megakaryocytes and platelets. So far, we have failed to observe immunoreactivity to tissue kallikrein in basophils. The presence of tissue kallikrein in extracts prepared from PMN leucocytes isolated from peripheral blood was demonstrated by immunodiffusion, dot-blotting and by radioimmunoassay. The kininogenase and amidase activity of the extracts resembled that of tissue kallikrein in being resistant to soya bean trypsin inhibitor and sensitive to trasylol. The amidase activity attributable to tissue kallikrein was completely inhibited by specific antisera.

High-molecular-weight kininogen is cleaved in active erythema multiforme

Erythema multiforme (EM) is an inflammatory disorder of the skin, which may include mucous membrane involvement, that features target (iris) lesions. Mediators specifically involved in EM are not well characterized; its pathogenesis remains enigmatic. In this study, evidence for participation of kinins in the pathophysiology of inflammation in EM was investigated by assessing cleavage of high-molecular-weight kininogen (HMWK) in plasma. These data were compared with analyses of plasmas from patients with serum sickness, chronic idiopathic urticaria/angioedema, and from normal control subjects. Patients with EM demonstrated significant levels of circulating cleaved HMWK in plasma during active disease ( p < 0.01), which declined during remission/recovery. Plasmas from patients with EM obtained during active disease also demonstrated significant levels of 94 kd C1 inhibitor ( p < 0.01) and C1 inhibitor-kallikrein complexes. Patients with serum sickness and chronic idiopathic urticaria/angioedema did not demonstrate these findings and did not differ from normal control subjects ( p = not significant). Although the kininogenase responsible for HMWK cleavage in EM has not been conclusively demonstrated, these findings suggest that HMWK cleavage resulted from activation of the contact system and that some of the manifestations of EM in selected patients may in part be accounted for by inflammatory and proinflammatory actions of kinins. Based on these preliminary findings, it will be important to establish whether or not HMWK cleavage in EM is a general finding in patients with this disorder. Further investigation is needed to characterize more clearly kininogenase activity and elucidate the possible role of kinin generation in EM.

Purification of Human Active Urinary Kallikrein: Comparative Inhibition Studies of Kininogenase and Amidolytic Activities

Large scale purification of human active urinary kallikrein is described. The final preparation was found homogeneous by means of SDS Page electrophoresis, amino acid composition and N-terminal analysis. The apparent molecular weight, determined on SDS Page electrophoresis, was 4.4 X 10(4). Comparative inhibition studies of the kininogenase and the amidase activities pointed out differences in the sensitivity of these two activities. Sodium inhibited amidase activity whereas kininogenase activity required the presence of this cation. In contrast, kininogenase activity was more sensitive to cadmium inhibition than amidase activity. Antibody against purified kallikrein did not completely inhibit amidase activity in crude urine. These discrepancies are consistent with the existence of several amidase activities in urine and also with possibly distinct catalytic sites on the same molecule, accordingly consideration of the methodology used appears very important when comparing results from different studies.

Glandular kallikrein-like enzyme in adrenal glands.

A kallikrein-like kininogenase was identified in the rat adrenal gland. Most of the enzyme was present in an inactive form, since pre-incubation with trypsin markedly increased kininogenase activity from 54.8 +/- 11.8 to 230 +/- 23.0 pg bradykinin/mg protein/min. Adrenal kininogenase was inhibited 90% by phenyl methyl sulfonyl fluoride, 92% by D-Phe-Phe-Arg-chloromethylketone, 91% by aprotinin, and only 15% by soybean trypsin inhibitor. Pre-incubation with antibodies against rat urinary kallikrein resulted in 85% inhibition. The apparent molecular weight of adrenal kininogenase on gel filtration chromatography was 33 Kd. The enzyme was strongly adsorbed to immobilized rat urinary kallikrein antibodies and required drastic conditions for elution. In canine adrenal glands, we found that there was no difference in the cortical and medullary distribution of active and inactive SBTI resistant kininogenase activity. We conclude that an enzyme which closely resembles glandular kallikrein is present in adrenal glands.

Urinary kallikrein excretion following chronic sinoaortic denervation in conscious dogs.

Urinary kallikrein excretion (UKE) was investigated in neurogenic hypertensive dogs for a period of 8 months. The animals were made hypertensive by sinoaortic denervation (SAD). Plasma catecholamine levels (PC), plasma renin activity (PRA), plasma aldosterone concentration (PAC) and urinary sodium excretion (UNa) were also measured. The onset of hypertension was associated with an increase in PC. UKE, measured by amidolytic and kininogenase activities, exhibited a very significant transient increase two and four weeks after SAD. Progressively, UKE significantly decreased below control values at the 16th and 32nd week. Since the month following SAD is characterized by an increase in sympathetic tone (as shown by high PC levels), the transient increase of UKE can be related to this high PC level; although this hypothesis is only supported by a positive relationship between these two parameters. The subsequent decrease in UKE appeared linked to diminished mineralocorticoid activity. Thus, the biphasic pattern of UKE observed in the study suggests that variations of UKE are more a consequence of hypertension than a pathogenic factor. Because UKE, which is of renal origin, is reduced at the end of the study period, this may also suggest possible renal dysfunctions in this model of hypertension.

Inhibition of human and rat tissue kallikreins by peptide analog antagonists of bradykinin.

Three representative bradykinin receptor antagonists have been examined for their capacity to inhibit purified human and rat urinary kallikreins. In an amidolytic assay, the Ki values for the inhibition of human urinary kallikrein were 0.5, 0.3, and 2.5 microM for B4307 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg), B4308 (Lys-Lys-Arg-Hyp-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg) and B3852 (Arg-Pro-Hyp-Gly-Phe-Ser-DPhe-Phe-Arg), respectively. B4308 and B3852 inhibited rat urinary kallikrein with Ki's of 1.0 and 5.5 microM, respectively. B4308 (0.4 to 1.6 microM) also inhibited the kininogenase activity of human urinary kallikrein on human low molecular weight kininogen in a dose-dependent fashion. These bradykinin receptor antagonists may inhibit the kallikrein-kinin system both by blocking the binding of kinins to B2 receptors and by inhibiting the cleavage of kinins from kininogens.

Identification of human lung mast cell kininogenase as tryptase and relevance of tryptase kininogenase activity

We have described previously the IgE-mediated release of kininogenase activity from purified human lung mast cells. Using supernatant fractions from mast cells stimulated with anti-IgE in the presence of deuterium oxide, we have purified this kininogenase to homogeneity by gel filtration and heparin-agarose chromatography and have demonstrated that it is identical to tryptase, the major neutral protease of human lung mast cells. Thus, tryptase and kininogenase activities co-chromatographed through both purification steps with equivalent yields. The final purified kininogenase was free of detectable chymotryptic and carboxypeptidase activities and was identified as tryptase on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition and inhibition profile. Three such preparations of tryptase were all capable of releasing kinin from each of two different preparations of purified, single-chain, human low molecular weight kininogen. Interestingly, kinin generation was optimal at pH 5.5 and was enhanced by heparin, which has been reported to stabilize tryptase. SDS-PAGE analysis of kininogen hydrolysis by tryptase revealed the formation of a diffusely stained region in the molecular weight range of 60,000–65,000, rather than a discrete heavy chain band. Under optimal conditions, the three tryptase preparations released 10–12 μg kinin/hr/mg but released only 2 μg kinin/hr/mg at pH7.2. HPLC analysis revealed that the kinin released was bradykinin. We conclude that the kininogenase activity from human lung mast cells is attributable to tryptase. The unique pH optimum of this reaction of a serine protease, however, raises doubts as to the physiologic significance of this activity.

Partial Purification of Plasma and Tissue Kallikreins in Psoriatic Epidermis

Human psoriatic scale extracts produced kinins from heated plasma (11.3 +/- 5.5 ng kinin/mg protein) and from purified low molecular weight (LMW) bovine kininogen (4.4 +/- 1.7 ng/mg). Sephacryl S-200 gel filtration of the extracts showed three peaks of kininogenase activity with Mr values of 90,000 (K-I), 65,000 (K-II), and 35,000 (K-III). Upon DEAE-Sepharose chromatography of the Sephacryl peaks, K-I activity was found in the nonadsorbed fraction and formed kinins only from heated plasma. Peak K-II activity was resolved into two peaks, K-IIa (in the nonadsorbed fraction), which formed kinins only from heated plasma, and K-IIb (in the adsorbed fraction), which formed kinins from both heated plasma and LMW bovine kininogen. K-III kininogenase activity appeared at the same position as K-IIb and also formed kinins from both substrates. Kininogenases K-I and K-IIa had the same Km value (0.3 mM) with Pro-Phe-Arg-p-nitroanilide(pNA), similar to that found with human plasma kallikrein. The Km value of K-IIb with Val-Leu-Arg-pNA (0.8 mM) was like that found for human salivary kallikrein, whereas K-III had a low affinity for this substrate. Like plasma kallikrein, K-I and K-IIa were inhibited by soybean trypsin inhibitor, but only weakly by aprotinin. In addition the kininogenase activity of both K-I and K-IIa was neutralized by adding antihuman prekallikrein immunoglobulin G (IgG). In contrast, K-IIb and K-III were strongly inhibited by aprotinin but not by soybean trypsin inhibitor, consistent with their being tissue kallikreins. It was confirmed that K-IIb and K-III shares antigenic determinant of urinary kallikrein.

Reduced urinary kallikrein in hypertensive diabetic patients

Renal kallikrein, an enzyme of the distal tubule acting on renal vasodilation through kinin liberation, may participate in the control of renal circulation and blood pressure. To study if impairment of kallikrein secretion can occur in diabetes, 40 nonhypertensive and 29 hypertensive diabetic patients were compared to 30 nondiabetic, nonhypertensive controls. Urinary kallikrein activity (UKA) was measured by its kininogenase activity. Preincubations with trypsin were performed to measure total kallikrein. Compared to UKA in controls [86 ± 9 μg lysylbradykinin (LBK, mean ± SEM) produced per minute of incubation], UKA was significantly reduced in both nonhypertensive (59 ± 8 μg LBK. min −1; p < 0.05) and hypertensive diabetics (26 ± 6 μg LBK. min −1; p < 0.001). The ratio of total/active urinary kallikrein was similar in diabetics and controls. From a multiple stepwise linear regression analysis, the prognostic independent variables for a reduced UKA in diabetic patients were creatinine clearance (Fisher's index: 34.64) and mean blood pressure (Fisher's index: 13.50). These results support the assumption that alterations of renal kallikrein release are associated with renal dysfunction and increased blood pressure in diabetic subjects.

Purification and characterization of a non-kallikrein arginine esterase from dog urine

A non-kallikrein arginine esterase (esterase I) has been purified from dog urine and characterized. The enzyme was purified by a three-step procedure, including ion exchange chromatography on DEAE-Sephacel, affinity chromatography on p-aminobenzamidine-Sepharose, and final gel filtration on Ultrogel AcA-54. The purified preparation gave three protein bands on polyacrylamide gel electrophoresis, all of which had esterolytic activity. The enzyme has a specific activity of 601 esterase units/mg protein. It has negligible kininogenase activity. Esterase I gave two closely migrating protein bands on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 34 000 and 33 300. Esterase I is a glycoprotein with a pH optimum of 9.5 and a p I of 4.62. The enzyme is strongly inhibited by a host of inhibitors including aprotinin, leupeptin, antipain, soybean trypsin inhibitor, lima bean trypsin inhibitor, and dPhe-Phe-Arg-chloromethyl ketone ( I 50 in the 10 −9 − 10 −8 M range). However, p-aminobenzamidine, Nα- p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I 50 values in the 10 −5 −10 −7 M range. The enzyme preferentially hydrolyzes Pro-Arg bonds. Among fluorogenic substrates used in this study, butyloxycarbonyl-Val-Pro-Arg-methylcoumarinamide (α-thrombin substrate) was found to be the best, with a K m of 1.7 μM and a k cat K m of 6.3 s · μM −1. However, esterase I does not convert fibrinogen to fibrin nor activate plasminogen to plasmin. Esterase I is immunologically distinct from dog urinary kallikrein, having no cross-reactivity with antibodies against dog kallikrein.

Short lasting increase in urinary specific kallikrein activity during long term administration of furosemide.

Furosemide was administered for seven days to normal rats. Urinary kallikrein excretion showed a biphasic response during the seven consecutive days of study. During the initial three days only the kininogenase activity showed a significant increase without any variation in the excretion of the immunoreactive kallikrein. The specific urinary kininogenase activity was therefore enhanced. After three days of furosemide administration, both the urinary kininogenase activity and urinary immunoreactive kallikrein were augmented. The urinary specific kininogenase activity was that time no more different when compared to the basal value. Considering the delay time of three days, this second part of the response could be a mineralocorticoid mediated effect. In this respect, kidney level of immunoreactive and kininogenase activity of kallikrein are also increased after seven days of furosemide administration. However the short lasting increase in urinary specific kininogenase activity observed during the initial three days is due to a change in the ratio active versus inactive kallikrein without any variation of total kallikrein. It is possible that this immediate response results of a direct effect of furosemide acting either on the preferential excretion of the active form or on the activation of the prokallikrein in the urine.

Renal Kallikrein Activity in Spontaneously Hypertensive Rats

To assess possible roles of the renal kallikrein-kinin system in the development of spontaneous hypertension, we determined daily excretion of urinary total and active kallikrein in 6-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats for up to 2 weeks. We also evaluated the effect of aprotinin, a reversible inhibitor of kallikrein and other serine proteases, on the development of hypertension in the 6-week-old SHR on ordinary intakes of sodium or on sodium loading with 1% NaCl for up to 2 weeks. Active kallikrein was determined by its kininogenase activity, and the generated kinins were radio-immunologically measured. Total kallikrein was also determined by measuring kininogenase activity after inactive kallikrein had been activated with trypsin (200 micrograms/ml). Urinary active kallikrein excretion was significantly reduced in 7-week-old SHR (1.5 +/- 0.2 microgram/day compared to 2.8 +/- 0.3 micrograms/day in WKY, P less than 0.05) and in 8-week-old SHR (1.6 +/- 0.2 microgram/day compared to 3.2 +/- 0.4 micrograms/day in WKY, P less than 0.01). Urinary total kallikrein excretion was also reduced in the 7- and 8-week-old SHR whereas the ratio of active to total kallikrein did not change. In addition, renal contents of total and active kallikrein were significantly lower in the 8-week-old SHR than in the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of kallikrein in cultures of adult renal cells

Cortical tubular cultures enriched with distal segments were prepared from the kidney of adult Fisher 344 rats bearing thyroid tumors. After two years of cultivation (20 passages) cell monolayers contained immunocytochemically detectable cytokeratin and kallikrein material in their cytoplasm. Furthermore cell pellets showed a true renal type of kininogenase activity corresponding to active kallikrein which ranged from 22.3 to 1.5, and total Kallikrein from 29.9 to 2.8 pg kinins/min per mg of protein.

Micropuncture localization of kallikrein secretion in the rat nephron

We have used free-flow micropuncture to study the tubular locus at which kallikrein enters the urine. Kallikrein was measured by a newly developed, very sensitive assay for kininogenase activity; active kallikrein was measured directly by this assay and total kallikrein after activation of inactive kallikrein. Kallikrein was readily detected in all of 17, late distal tubular fluid-samples. In contrast, kallikrein was too low to detect in 15 of 17 proximal or in 11 of 14 early distal tubular fluid samples. Calculations indicate that less than 10% of urinary kallikrein could have derived from filtration or from proximal secretion of kallikrein. We conclude that urinary kallikrein enters the urine via secretion in the distal tubule. Filtration or proximal secretion of kallikrein does not contribute significantly to urinary kallikrein excretion.

Kallikrein (kininogenase) in the mouse nephron: effect of dietary potassium.

Kininogenase activity of kallikrein was measured in microdissected mouse nephron segments using kininogen from dog plasma and a radioimmunoassay for bradykinin. When single nephron segments were examined, results showed a large scatter. This was found to be due to heterogeneity of distal convoluted tubules (DCT) from different nephrons, since replicate measurements in pools of DCT structures did not show this degree of variation. Nearly 20% of activity was accessible to extracellular substrate when freshly dissected segments were incubated in isoosmotic media. Freezing and thawing which markedly releases activity of intracellular enzymes, did not significantly elevate kininogenase activity. On the other hand deoxycholate and trypsin treatment increased tubular kininogenase activity in an additive fashion. A detailed analysis of microdissected tubule fragments revealed that kallikrein is concentrated in late distal convoluted tubule before entering a branching point (connecting tubule). In contrast initial portions of distal convoluted tubules and cortical collecting tubules contained only little kallikrein activity. Potassium rich diet increased basal and total activity 5-fold, when compared to a potassium poor diet.

Detection of tissue kallikrein in the bronchoalveolar lavage fluid of asthmatic subjects.

Kininogenase activity was detected by cleavage of radiolabeled substrate (125I-high molecular weight kininogen [HMWK]) in 22 of 24 bronchoalveolar lavage (BAL) fluid samples from 17 asthmatics who either responded to aerosolized allergen challenge or had symptoms of active asthma. In contrast, six of seven normal controls lacked enzymatic activity. Levels of free immunoreactive kinin found in BAL fluid correlated with the presence of kininogenase activity (P = 0.002). The cleavage pattern of 125I-HMWK by the BAL fluid kininogenase (a dominant 65,000-mol wt fragment), and synthetic inhibitor profile (phe-phe-arg-CH2Cl and phenylmethylsulfonyl fluoride) were compatible with a tissue kallikrein. Peak kininogenase activity eluted at an apparent molecular weight of 20,000-34,000 by HPLC gel filtration. Its antigenic identity was established by immunoblotting with anti-human urinary kallikrein antibody and its activity was inhibited by this antibody. Lysylbradykinin was generated during incubation of fractionated BAL fluid and purified HMWK, the characteristic cleavage product of the tissue kallikreins. We conclude that elevated amounts of tissue kallikrein and kinin are present in the bronchoalveolar spaces of asthmatic subjects. Kinin generation may contribute to the asthmatic response directly through edema formation and smooth muscle contraction and by augmenting release and/or production of preformed (histamine) and secondary mediators such as leukotrienes and platelet-activating factor.

Isolation, characterization, and localization of antigen gamma, a serine proteinase of the "kallikrein-family" in the rat submandibular gland.

A trypsin-like serine proteinase, antigen gamma, immunologically partially identical to glandular kallikrein when run against anti-rat glandular kallikrein antiserum in immunoelectrophoresis, was purified from the rat submandibular gland. The enzyme was purified by a two-step chromatography procedure, ionexchange chromatography followed by gel filtration. The criteria for purity were one band in SDS-polyacrylamide gel electrophoresis and in immunoelectrophoresis, respectively. Antigen gamma had a molecular mass of 25,000 Da and consisted of two polypeptide chains with molecular masses of 14,000 and 11,000 Da. The preparation contained several isoenzymes with pI ranging from 4.1 to 4.5. The enzyme showed high specific enzyme activity against the substrate D-valyl-L-leucyl-L-arginine-4-nitroanilide (S-2266), some trypsin-like and kininogenase activity, but no angiotensin converting enzyme, kininase, or tonin activity. Amidolytic activity was increased and stabilized by the presence of detergent in the assay buffer. The pH-optimum of antigen gamma amidolytic activity was about 10. Antigen gamma was inhibited by SBTI and PMSF, whereas aprotinin had to be added in a more than 100 times higher concentration than for glandular kallikrein. The binding pattern of antigen gamma to plasma proteins was different from that of tonin and glandular kallikrein. Antiserum against antigen gamma was raised in rabbits and characterized against rat submandibular gland homogenate. Immunohistochemistry showed antigen gamma in the secretory granules of the submandibular gland granular tubular cells but only adhering to the luminal cell wall in the striated and main excretory ducts. Antigen gamma was not detected in the sublingual or parotid gland or in the kidney. Antigen gamma was demonstrated by immunoelectrophoresis in rat submandibular gland saliva. The concentration was higher in sympathetically than in parasympathetically induced secretion.

Specificity of the amidase and kininogenase methods for the determination of rat urinary kallikrein.

The specificity of the amidase and kininogenase methods for determining rat urinary kallikrein was studied. Male and female rat urine was employed. Esterase A1, A2 and kallikrein were separated by DEAE-Sephadex A-50 chromatography. Esterase A1 showed no amidase activity towards the substrate H-D-Val-Leu-Arg-p-nitroanilide. In contrast, esterase A2 and kallikrein attacked the substrate, and the activity of kallikrein was especially inhibited by aprotinin, while esterase A2 was more sensitive to soybean trypsin inhibitor. Esterase A1 did not show kininogenase activity, whereas esterase A2 showed this activity, but only towards the dog plasma substrate. Kallikrein possessed kininogenase activity towards both dog and rat plasma kininogen. We believe that the most specific method for measuring rat urinary kallikrein activity is the kininogenase method using partially purified rat plasma kininogen.

Urinary kallikrein excretion during inhibition of endogenous angiotensin II in the pig.

This study was performed to assess the possible contribution of endogenous angiotensin II (AII) to the regulation of urinary kallikrein excretion. The AII antagonist saralasin or the saline vehicle was infused into the aorta above the renal arteries of pigs under halothane-O2/N2O anaesthesia. Systemic and renal functional parameters were followed for 140 min and during stimulation of the reninangiotensin system by haemorrhage. Urinary kallikrein excretion, determined as kininogenase activity, was increased immediately upon both initiation and termination of the 2 h saralasin infusion into pigs not subjected to haemorrhage. Renal cortical blood flow (RCBF) was maintained, in both saline and saralasin-treated animals at blood pressures as low as 70 mm Hg, while glomerular filtration rate was dissociated during saralasin infusion. As long as RCBF was maintained, urinary kallikrein excretion rate was elevated during the progressive hypotension in both saline and saralasin-treated animals. These findings confirm a close relationship between the maintenance of RCBF and increased activity of the kallikrein-kinin system whether or not AII is antagonized, and indicate that during haemorrhage the kallikrein-kinin system is stimulated by a mechanism not involving AII.