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... Rat plasma contained far less of this kininogen than dog plasma. Similar findings were made by Jacobsen (1966b). Therefore rat plasma was found to be an unsuitable source for preparation of kininogen. ...
1. Rat kidneys which were perfused with saline contained both kininogenase (KGA) and kininase activity. These activities were separated by gel filtration on a Sephadex G-100 column. The kininase activity was excluded from the column whereas the KGA activity was retained. Kidney KGA activity was primarily found in the sedimentable fraction of the homogenate.2. The kidney KGA activity was compared with the urinary KGA activity, and the following properties were found to be the same: molecular dimension, pH optimum, effect of inhibitors, and ability to liberate kinins from kininogens.3. A urinary sample collected over 24 h contained about 8 times the KGA activity found in the corresponding kidneys at the end of the collection period. The urine: kidney ratio for alkaline phosphatase was about 0.01.4. The ability of kidney and urinary samples to hydrolyse N-alpha-benzoyl-L-arginine ethyl ester (BAEE) at pH 8.5 paralleled the KGA activity.
... infusion through the jugular vein ol conscious rats in which the indwelling cannula had been inserted under ether anaesthesia 24 h earlier. The drugs were administered in the doses and following the schedules indicated inTable 3Jacobsen, 1966). The samples were centrifuged at 800 £ at 4°C and the plasma obtained was assayed for kinin-and prostaglandin-like activity by the cascade superfusion procedure (Naylor, 1977). ...
Uterine blood flow in ovariectomized rats was measured by means of radioactive microspheres. Blood flow was increased from 55 ml min-1 100 g-1 by treatment (i.v.) with 0.5 microgram oestradiol kg-1 and reached 680 ml min-1 100 g-1 within 60 min. This oestrogen-induced increase of blood flow was reduced significantly by pretreatment with mepyramine (a histamine H1-receptor antagonist), cellulose sulphate (a kininogen-depleting agent) and aprotinin (a kininogenase inhibitor). Cimetidine (a histamine H2-receptor antagonist), kallikrein (kininogenase enzyme) and atropine (an anticholinergic drug) had no effect on the increased uterine blood flow. Indomethacin and AH 7170, which inhibit the formation of prostaglandins, also caused a lower increase in uterine blood flow. None of the pretreatments fully inhibited the oestrogen-induced increase in blood flow, suggesting that more than one mediator may be involved.
Evidence has been accumulating for some time that different glands of several mammalian species contain substances of protein nature which can have powerful vasodepressor effects. These substances can be readily extracted from the glandular tissues and appear in an active form in their secretions. It was only natural that these findings should lead to speculation, some of it quite early, about their possible physiological role. Thus, the first ideas about what are now known as the kinin systems were propounded under a different name. In their earliest definitive work, Kraut et al. (1930) had shown that extracts of pancreas and pancreatic juice owed their vasodepressor activity to a material of protein nature which they called kallikrein (see p. 2). Having found a similar substance in extracts of blood and urine, they put forward the idea that they were dealing with a new vasodilator hormone which was secreted by the pancreas, circulated in the blood stream in an inactive form, and then excreted. Physiological and clinical interest in this notion waned, but Werle and his colleagues carried on to show that, in part at least, the action of kallikrein was due to the release of a hypotensive substance from plasma (Werle et al., 1937; Werle and Berek, 1948).
Indirect evidence has been provided for the presence of 3 kininogen fractions: The average amounts of kinin released by rat plasma kallikrein (1.5 μg/ml plasma, S. E. M. = 0.03) and by rat urine kallikrein (1.4 μg/ml plasma, S. E. M. = 0.03) in 7 plasma batches corresponding to a total of 90 rats, when added up, significantly exceeded the total kininogen (2.0 μg/ml plasma, S. E. M. = 0.04). Methods and materials were as described by BRISEID, DYRUD & ÖIE (1970). It is suggested that plasma kallikrein released kinin from 2 kininogen fractions, S1″ and S1″, and that urine kallikrein released kinin from 2 kininogen fractions, S1″ and S2. Repeated incubation with each of the kininogenase preparations used did not increase the yield of kinin. Soybean trypsin inhibitor did not reduce the amount of kinin released by urine kallikrein; the plasma kallikrein, however, was strongly inhibited. In control experiments leucine aminopeptidase transformed kallidin to bradykinin, but did not increase the kinin activity of the urine kallikrein incubates.
High-molecular-weight kininogen was purified to apparent homogeneity from Wistar rat plasma by a two-steps chromatographic procedure. 3 mg of kininogen were obtained from 205 ml of plasma. The purified high-Mr kininogen had a bradykinin content of 10.2 micrograms bradykinin equivalents/mg protein. Under denatured and reduced conditions it gave a single band on polyacrylamide gel electrophoresis corresponding to an apparent molecular mass of 110 kDa. Antibodies obtained against rat high-Mr kininogen gave a single precipitation line when tested against rat plasma in double immunodiffusion and in crossed immunoelectrophoresis. Although rat high-Mr kininogen possesses physicochemical properties (molecular mass, kinin content per molecule and amino acid composition) similar to human high-Mr kininogen, its antibodies do not cross-react with human, monkey or rabbit plasma, indicating major interspecies differences in the structure of the molecule. Immunoreactive kininogen of Wistar rats was identical to that of Brown Norway rats from a strain bred in Orleans, France (BN/Orl). However, plasma from a strain of Brown Norway rats bred in Leuven, Belgium (BN/Kat), reported to be deficient in a kinin precursor (Damas, J. and Adam, A. (1980) Experientia 36, 586-587), did not contain immunoreactive material discernible by double immunodiffusion or crossed immunoelectrophoresis.
1. The time course of oedema formation in rats caused by injection of carrageenin into the paw was followed for 5.5 hours. Intact or adrenalectomized rats which had previously been injected with ellagic acid or saliva to reduce considerably the concentration of blood kininogens, or with methysergide to antagonize 5-hydroxytryptamine (5-HT) showed a reduced inflammatory response. It was concluded that kinins and 5-HT contributed significantly to oedema formation during this period.2. Mepyramine alone had no effect on oedema formation, but in combination with ellagic acid treatment, with or without methysergide, it caused a reduction suggesting that histamine played a minor role in oedema formation during the first 3 hours.3. Vascular permeability studies indicated that injection of ellagic acid did not interfere with the normal responses in skin to intradermal injections of histamine, 5-HT, bradykinin or compound 48/80. Mepyramine and methysergide, at the doses used in the carrageenin experiments, completely antagonized histamine and 5-HT, respectively, and did not affect the skin responses of bradykinin.4. Treatment in vivo with ellagic acid or rat saliva was equally effective in reducing plasma kininogen concentrations by an amount equivalent to more than 10 times the quantity of substrate 1 measured by Gautvik & Rugstad (1967).5. Rat saliva, but not ellagic acid, lowered complement levels by approximately 20%.
1 (-)-Adrenaline lowered the kininogen content and transitorily elevated the fibrinolytic activity of plasma following intravenous injection into the rat. Its effect on kininogen increased when administered by intravenous infusion.2 Although less effective, (-)-noradrenaline had a similar action to adrenaline; (+/-)-isoprenaline was inactive and failed to inhibit the effect of adrenaline.3 The effect of adrenaline on kininogen could be reproduced in vitro by incubation of whole blood, but not cell-free plasma, with the catecholamine for 5 min at 37 degrees C.4 Propranolol or phenoxybenzamine, as well as heparin or acetylsalicylic acid (aspirin), blocked the reduction of rat blood kininogen by adrenaline in vivo and in vitro.
1. A method is described for determination of kininogen 1 (substrate mainly for plasma kallikrein) and kininogen 2 (substrate for glandular kallikrein) independently in cat plasma.2. In anaesthetized cats the arterial inflow to, and venous outflow from, the submandibular salivary gland were isolated: a roller pump giving constant volume inflow was interposed in the arterial circuit.3. Venous blood was collected at rest, during and after stimulation of the chorda tympani, and its content of kininogens 1 and 2 were estimated. Kininogen 2 was reduced up to 60% by chorda stimulation, whereas the level of kininogen 1 was unchanged.4. On close arterial infusion of bradykinin or histamine in amounts which produce large vascular effects, including increased capillary permeability, the venous blood levels of both kininogens 1 and 2 were unchanged.5. It is concluded that the selective loss of kininogen 2 on chorda stimulation results from the release of kallikrein into the tissue spaces and reflects the extent of kinin formation within the gland.
Focused microwave irradiation was employed to stabilize endogenous whole rat brain bradykinin levels prior to a simple extraction procedure. Skull microwave exposure (2450 MHz, 3.8 kW., 2.45 sec) resulted in inactivation to less than 5% of control of whole brain kallikrein and kininase activity. Using this adequate exposure duration whole rat brain kinin levels as measured by a sensitive radioimmunoassay were approximately 0.6 pmol/g (wet weight). Further purification of irradiated brain extracts using HPLC revealed that immunoreactive kinin eluted as a single peak that co-chromatographed with authentic bradykinin. Microwave fixation duration of 1.25 sec yielded greatly increased levels of immunoreactive kinin which following HPLC purification eluted in two peaks, corresponding to authentic bradykinin and T-kinin, respectively. The tissue injury resulting from incomplete microwave fixation resulted in the release of kinins. This excess immunoreactive kinin may be derived from cerebral blood, since the predominant form of kinin-generating protein in plasma is T-kininogen.
The kininase activity and the level of the two kininogens, substrate 1 and 2, have been determined in plasmas from cats, hamsters and guinea-pigs. Kininase activity was found to be far more pronounced in the latter type of plasma than in the former ones. The efficiency of di-sodium edetate and phenanthroline as kininase inhibitor was also evaluated in the three plasma types. In plasma from cat and hamster, human plasma kallikrein was found to release kinins from the socalled substrate 1 only, whereas in guinea-pig plasma the same agent released kinin from both types of kininogens. The animal's own saliva acted only upon substrate 2 in plasma from cat and guinea-pig, whereas it induced kinin formation from both substrates in plasma from hamster. Ellagic acid and glass contact released kinins from substrate 1 only in all three plasma types. Intravenous injection in hamsters and guinea-pigs of a buffered solution of ellagic acid, caused pronounced effects on blood pressure and respiration. On repeated injections these effects subsided. After four such injections the concentration of substrate 1 in arterial blood samples from hamsters was found to be very low (varying from 0 to 20 %). The level of substrate 2 was, however, unchanged. The level of substrate 1 reached 83 % of the initial value after 24 hrs.
T-kinin, a previously undescribed peptide containing bradykinin, has been isolated following treatment of rat plasma with trypsin (1 mg/ml). The liberated T-kinin, which contracts the rat uterus, was isolated by procedures including OM-cellulose, Biogel P-4 and reverse-phase high-performance liquid chromatography. The final material had a single N-terminal isoleucine and was shown by amino acid analysis and sequence determination to have the structure of the undecapeptide Ile-Ser-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (isoleucyl-seryl-bradykinin). The relationships of the protein from which T-kinin is cleaved (T-kininogen) to other known kininogens is discussed.
The kallikrein-kininogens-kinins system has been investigated in the brown Norway rat. In this breed the stores of kininogens in the plasma are reduced and the plasma kallikrein-like activity appears to be absent both in vivo and in vitro.
A high molecular weight kininogen has been isolated from rat plasma and purified. At each preparative step the kininogen concentration and purity were monitored by assay on the perfused isolated rat uterus in terms of bradykinin equivalents formed per mg protein following incubation of the plasma fractions with rodent acid protease for 24 hours at 37 degrees and pH 4.0. Kinin formation by crystalline trypsin and human pancreatic kallikrein also was compared. Citrated rat plasma first was precipitated with 43% ammonium sulfate. The kininogen fractions then were subjected to a series of gel filtration ion exchange chromatographic columns that included G-200 Sephadex, G-200: G-100 Sephadex interconnected columns, DEAE-A50 Sephadex, and hydroxylapatite. The kininogen fractions finally were subjected to preparative polyacrylamide gel electrophoresis, resulting in a final purification of 92.9-fold compared to the initial rat plasma. A single major kininogen protein band and a minor band of protein impurity were obtained on disc gel electrophoresis. Only the pancreatic kallikrein did not form kinin from this purified kininogen. The apparent molecular weight was estimated by SDS polyacrylamide gel technique to be 110,000.
The effects of ouabain, ATP, and vanadate on palytoxin induction of ion channels were examined with the aim of elucidating the role of Na,K-ATPase in palytoxin action. Palytoxin-induced membrane depolarization of crayfish giant axons and single channel currents of frog erythrocytes and mouse neuroblastoma N1E-115 cells were examined using the intracellular microelectrode and patch-clamp techniques. External application of palytoxin in nanomolar concentrations induced depolarization in the crayfish giant axons, and the depolarization was inhibited by pretreatment of the axon with ouabain (10 μM). Internally perfused axons were less sensitive to palytoxin unless ATP (6 mM) was added internally. In patch-clamp experiments, picomolar palytoxin in the patch electrode induced single channels in both cell-attached and inside-out patches of erythrocytes and neuroblastoma cells. The induced channels had a conductance of about 10 pS, reversed near 0 mV in physiological saline solution, and was permeable to Na+, K+, Cs+, and NH
inf4sup+, but not to choline. Single channel activities induced by palytoxin were inhibited by ouabain (10 μM) and vanadate (1 mM), but promoted by ATP (1 mM). The modulating effects of ouabain, vanadate, and ATP on palytoxin action suggest that the Na,K-ATPase is involved in the induction of single channels by palytoxin. Palytoxin-induced and ouabain-inhibitable single channels were observed in planar lipid bilayer incorporated with purified Na,K-ATPase. The results indicate that an interaction between palytoxin and Na, K-ATPase leads to opening of a 10-pS ion channel. They further raise the possibility that a channel structure may exist in the sodium pump which is uncovered by the action of palytoxin.
Two species of T-kininogen which release T-kinin (Ile-Ser-bradykinin) have been purified from plasma of rats treated with Freund's complete adjuvant. The molecular weight was estimated to be 69,000 for either T-kininogen I and II by SDS-polyacrylamide gel electrophoresis. Trypsin released one mole of T-kinin from one mole of either T-kininogen, but glandular kallikrein, including rat urinary and rat submandibular gland kallikreins and human urinary kallikrein, did not release any kinin from T-kininogens. Cathepsin D, which was purified from rat liver, released T-kinin from T-kininogens at pH 4.0. These results indicate that rat plasma contains two types of T-kininogen which differ from high molecular weight and low molecular weight kininogens.
Purification and further characterization of a kinin-forming acid protease in a mouse fibroblast L929 stationary cell culture line was carried out. Supernatants of dialyzed fibroblast homogenates digested denatured hemoglobin at pH 4.0. The supernatant was fractionated on a G-200 Sephadex column, hydroxylapatite column and finally on a DEAE-A50 Sephadex ion exchange column. A 9.4 fold purification was achieved with a 13.8% yield. The enzyme had a specific activity of 2062 ng kinin per mg protein when measured on a purified rat kininogen using the isolated rat uterus as the bioassay tissue. The protease had a pH optimum of 3.8-4.0. Molecular weights of the enzyme and substrate estimated on a G-200 Sephadex column were 39,000 and 110,000 respectively. Kinin formation was a function of both incubation time and enzyme concentration. Protease activity was localized primarily in the 10,000 g supernatant cell fraction (61.5%) with the 1500 g precipitate cell fraction containing 38.5% of the activity.
Original works written in French, which deal with the pharmacological action or the pathological role of the plasma kinins, are summarized below. I shall mention only review articles based on papers published in other languages: Berde (1961), Lecomte and Troquet (1962 a), Stürmer (1963), Rocha E Silva (1963), Troquet and Lecomte (1964), and Foussard-Blanpin (1965).
Low molecular weight (LMW) kininogen was purified 70-fold with a 16% yield from fresh rat plasma by DEAE-Sephadex chromatography, ammonium sulfate precipitation, Sephadex G-200 gel filtration, SP-Sephadex chromatography, CM-cellulose chromatography, and Sephadex G-200 gel filtration. Ferguson plots of polyacrylamide gel electrophoretic patterns revealed four bands with relative molecular weights of 64,000, 123,500, 252,436 and 357,900 (ratio of 1:2:4:6). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis provided a single protein band with a molecular weight of 72,000, suggesting that the four kininogen bands had been caused by the aggregation of a single oligomeric protein. The purified LMW rat kininogen Fraction B (3.9 μg bradykinin/mg) was used to elicit an antiserum in the rabbit. Monospecificity of the antiserum was demonstrated by immunoelec-trophoresis (Laurell rocket and Grabar methods) and, thus, the homogeneity of the kininogen was also. The purified kininogen (both Fractions A and B) formed kinin with human urinary kallikrein, rat urinary kallikrein and hog pancreatic kallikrein. Murphy-Sturm lymphosarcoma acid protease also formed kinin when incubated with the kininogen at pH 3.0. The isoelectric point for both fractions was at pH 4.3. Amino acid analyses showed the two kininogen fractions to be rich in acidic amino acids and to have a total carbohydrate content of 8.5% consisting of galactose (1.2 to 1.5%), mannose (1.9 to 2.1%), N-acetylglucosamine (4.3 to 5.1%), N-acetylgalactosamine (0.3%), and sialic acid (0.68%).
Many reports describing procedures for purifying the components of the kallikrein-kininogen-kinin system have appeared since Frey and co-workers (Frey, 1926; Frey and Kraut, 1926) initiated the work on urinary kallikrein which led to the discovery of this system. However, not until the advent of high-resolution separation methods in the last twelve years has much progress been made toward the isolation of these proteins in pure and biologically active form. The older methods, such as the Cohn alcohol fractional precipitation of plasma proteins (Cohn et al., 1946), tend to be nonspecific by present-day standards and thus inefficient, and to cause denaturation and hence loss of activity. The newer methods usually employ mild conditions of pH, temperature, and solvent, and so favor the retention of biological activity. Such methods also embody a continuous countercurrent principle which magnifies small differences between closely similar compounds, thus making possible high resolution and, as a corollary, high recovery. Outstanding examples are ordinary and recycling gel filtration; gradient elution chromatography on columns of ion exchangers and hydroxyapatite; preparative polyacrylamide gel electrophoresis and isoelectric focusing; and ultracentrifugation in density gradients. Reviews of the theory and applications of these techniques appear in alternate years in Analytical Chemistry [see Anal. Chem. 40 no. 5, IR-620R (1968)]. The reader is referred also to the useful book on chromatography edited by Heftmann (1967). Future progress in the purification of proteins and other macromolecules will probably lie chiefly in the further development of affinity adsorption methods (Cuatrecasas et al., 1968). Examples are the use of immunoadsorbents for the purification of antigens and antibodies and the use of inhibitor and enzyme columns for the purification of enzymes and inhibitors respectively (Fritz et al., 1967 b, c, 1968, 1969 a, b).
The earliest citations of a hypotensive substance (urohypotensine) resembling kallikrein appeared in the publications of Abelous and Bardier (1908, 1909). Later, similar hypotensive activity was observed by Pribram and Hernheiser (1920) in urine and by Migay and Petroff (1925) in pancreatic juice. About 50 years ago, attempts by Frey and Kraut to locate the source of a hypotensive macromolecule (F-Stoff) which they had characterized in urine (Frey and Kraut, 1928) and in serum (Kraut et al., 1928) led them to the discovery of a similar substance in a pancreatic cyst (Frey et al., 1930). Mammalian pancreas was subsequently found to contain such large amounts of F-Stoff that it was believed to be the site of origin of the hypotensive activity observed by them in urine and blood. This new substance was called “Kreislaufhormon” because of its marked vascular effects. F-Stoff was renamed “kallikrein,” the term being derived from the Greek word Kallikreas meaning pancreas. Later experiments indicated that pancreatic kallikrein was primarily released into the pancreatic juice in a precursor form which was readily activated by duodenal enterokinase (Frey and Werle, 1933; Werle, 1934). This finding spurred Werle and colleagues to search for this enzyme in other glandular tissues. Investigation of salivary tissue resulted in the identification of kallikrein in the submandibular glands of a number of mammals.
The presence of two kininogens has been shown in human, dog, guinea pig, rabbit and rat plasma by Jacobsen (1–3), in horse plasma by Henriques et al.(4), and in bovine plasma by Yano et al. (5). Pierce and Webster (6) isolated two low molecular weight (LMW) human kininogens with the same molecular weight of about 50,000 but with their kinin sequences carboxyl-terminal in one case and internal in the other. After that, two kininogens of different molecular weight were isolated from human plasma by Jacobsen and Kritz (7). LMW bovine kininogen (kininogen-II) has been highly purified by the groups of Habermann (8) and of Suzuki (9), and it was found by us that the kinin segment, which was thought to lie in the central part of LMW kininogen molecule, was released by trypsin, snake venom kininogenase or hog pancreatic kallikrein but not by glass-or casein-activated bovine plasma kallikrein. High molecular weight (HMW) kininogen (kininogen-I), a sensitive substrate of bovine plasma kallikrein, was isolated from bovine plasma by Yano et al.(10), and it was found that there is a methionyl-lysyl-bradykinin sequence located both at the carboxyl terminus and inside of the polypeptide chain of bovine HMW kininogen (11).
Studies on the kinin system were initiated some 50 years ago by Frey and coworkers (Frey and Kraut, 1928; Kraut et al., 1928; Kraut and Werle, 1930) who showed that urine contains a hypotensive substance. These studies confirmed an earlier observation by Abelous and Bradier (1909) that the injection of urine into dogs was associated with lowering of the systemic blood pressure. Kraut and Werle (1930) named the hypotensive substance, kallikrein, since they were able to isolate large amounts of the hypotensive substance from the pancreas. It was later found that the mixture of plasma with urinary kallikrein caused contraction of an isolated segment of guinea pig ileum; this activity being initially called “darmkontrahierende Substanz” (Werle et al., 1937). Subsequently, the name kallidin was substituted for darmkontrahierende Substanz and its precursor form was designated as kallidinogen (Werle and Berek, 1948). Concurrently, Rocha e Silva et al. (1949) discovered that the incubation of trypsin or the snake venom of Bothrops jararaca with a pseudoglobulin fraction of plasma resulted in the formation of a potent vasodilator and smooth-muscle-stimulating substance. Because the guinea pig ileum responded slowly to the substance, compared with histamine or acetylcholine, the spasmogen was designated bradykinin.
The kallikreins have been defined as endogenous enzymes which rapidly and specifically liberate a kinin from plasma (Appendix on Nomenclature). Perhaps more comprehensively they might be defined as those proteolytic enzymes of animal origin which release a kinin from kininogen but, as far as is known, do not readily cleave peptide bonds in other proteins. The term kininogenase is a general one which has been applied to any enzyme that liberates a kinin from an inactive protein substrate. However, not all authors agree with this terminology, preferring to call all these enzymes kininogenins since to them the former expression implies the destruction of kininogen and the latter the formation (genesis) of active kinins (Rocha e Silva, 1962, 1963, 1965, and personal communication). For the purpose of this chapter the term kallikrein will be restricted to mammalian and avian kallikreins and will not include those enzymes found in snake venom, which may also be kallikreins (Iwanaga et al., 1965).
The discovery of kininogen dates back to the observation made by Werle et al. in 1937 that kallikrein by itself fails to contract isolated smooth muscular orrgans; addition of serum was necessary. Werle had already excluded a possible “cofactor” function of serum—the relationship between kallikrein and serum resembled that between enzyme and substrate. The second observation was made by Rocha e Silva et al. (1949) a decade later: they described a gut-stimulating principle in blood after application of snake venom in vivo or in vitro. From the historical point of view it is remarkable that both independent groups detected kinin activity only in combination with the kinin precursor. This applies to all the progress made in exploring the kinin system. Better knowledge of biochemistry and pharmacology of kinins contributed to our understanding of kininogen and vice versa. The definition of the term kininogen is unambiguous. It covers every protein that yields kinin(s) on incubation with suitable enzymes.
A differential assay method for high molecular weight (HMW) and low molecular weight (LMW) kininogens was developed. Plasma from human or rat was divided into three portions for the following purposes. For total kininogen, plasma was pretreated at pH 2.0 and bradykinin released by trypsin was separated and assayed on rat uterus. For HMW kininogen, plasma was incubated with glass powder in the presence of o-phenanthroline (a kininase inhibitor). The released bradykinin was separated and assayed. For LMW kininogen, plasma was incubated with glass powder in the absence of o-phenanthroline. The HMW kininogen-depleted plasma was treated in the same way as for the total kininogen assay. The HMW, LMW and total kininogen levels were as follows: 0.98 ± 0.05, 3.08 ± 0.09, 4.11 ± 0.10 for human and 0.96 ± 0.06, 0.94 ± 0.16, 1.93 ± 0.11 μg bradykinin equivalent/ml plasma for rat. From the results using heated human plasma (60°C, 1 hr), 10% of normal plasma was required to obtain full formation of kinin from HMW kininogen by 30 min incubation with this method.
One of the most important causes of the phenomenon of oncolysis by the Clostridium strain M55 may be the release of kininases by the vegatative rods into the environment, resulting in a kinin deficiency in the capillaries of the tumor.
Experiments with ellagic acid have been successful in potentiating the deficiency of kinin produced by the spores of Clostridium M55 in malignant tissue, thereby improving oncolysis.
Ellagic acid activates the Hageman factor and liberates kinin from the plasma. Solid Yoshida tumors in rats that were treated with ellagic acid and spores showed definite recognizable lysis. The application of spores alone produced no lytic effects.
The results open the possibility of successfully treating humans with Clostridium M55 and simultaneously with agents that have an influence on the kinin forming system.
Physiological variation of kinin precursor (kininogen), in the plasma of animals not subjected to drugs or other treatment has been comparatively little studied. The present note provides information on the influence of age and sex on total, as well as on the portion of plasma bradykininogen which is mobilized in rats receiving cellulose sulfate, a kinin-releasing agent (Rothschild and Gascon, 1966).
Methods were developed for determining the kininogen fractions, kininase and prekallikrein. The plasma prekallikrein was activated by 20 % (v/v) acetone for about 17 hours (20–24°). Urine kallikrein was prepared by dialysis of urine against running tap water for about 24 hours. Kininase activity was eliminated in plasma, plasma kallikrein and urine kallikrein by incubation at 37° with EDTA-2Na (1.0 × 102 M) for about 24 hours. Kinin assays were carried out on the isolated rat uterus. Released kinin was calculated as μg bradykinin/ml plasma. The total kininogen, whether determined by activation with acetone (16 % v/v for not less than 5 hours) and subsequent incubation with plasma kallikrein, by incubation with plasma kallikrein and then urine kallikrein, or by incubation with acetone (20 % v/v for 17 hours) and subsequent evaporation of the acetone, was found to be the same, 2.0 μg/ml plasma as an average value of 7 plasma batches corresponding to a total of 90 rats (S. D. = 0.09). The average values of kinin released by incubation with plasma kallikrein and by urine kallikrein were 1.5 μg/ml and 1.4 μg/ml plasma respectively with S. D. values of 0.11 and 0.06 respectively. The procedures for kininase and for prekallikrein determinations corresponded closely to previously published methods for estimation of the same parameters in human plasma (RINVIK, DYRUD & BRISEID 1966; BRISEID, DYRUD & ARNTZEN 1968).
The location of the kinin moiety in high molecular weight (HMW) bovine kininogen (kininogen-I), a sensitive substrate for serum kallikrein, was investigated. Treatment with carboxypeptidase B destroyed about one-half of the initial kinin-yielding ability of kininogen-I, and selective cleavage of the methionyl peptide bonds in kininogen-I with cyanogen bromide liberated free kallidin (lysyl-bradykinin) and an inactive kallidin-containing peptide. These results show that there is a methionyl-lysyl-bradykinin sequence located both at the carboxyl terminus and inside of the polypeptide chain of bovine kininogen-I.
Purification and further characterization was carried out on a kinin-forming acid protease isolated from a rodent fibroblast cell line L-929 grown in stationary cell culture (N. Back and R. Steger,). The cells, cultured in minimal essential medium containing 10% fetal calf serum and 0.4% lactalbumin, were homogenized, the homogenate dialyzed for 18 hr against 0.01 M phosphate buffer at pH 6.8 in 0.1 M NaCl and 1.0 mM EDTA, and centrifuged at 10000 rpm for 45 min. The supernatant, which digested denatured hemoglobin at pH 4.0, was fractionated first on a G-200 Sephadex column. Kinin-forming activity, compared with that of the supernatant on an isolated perfused rat uterus preparation, was identified in fractions 25–40 when incubated for 24 hr at pH 4.0 with rat plasma kininogen substrate. The active fractions were pooled and purified further on a hydroxy-patite column. Treatment of the active fractions with 5 mM cysteine increased the activity 2-fold. Final purification was carried out on a DEAE-A50 Sephadex ion exchange column. The purification factor, compared to the initial supernatant, was 9.4 with a 13.8% yield and a specific activity of 2062.5 ng kinin per mg protein. Dialyzed and centrifuged rat plasma fractionated on a DEAE-A50 Sephadex column initially yielded two apparent kininogen species which resolved into a single major molecular species following passage through a G-100 Sephadex column. The purified enzyme and substrate preparations were used to establish the optimum kinin-forming activity at pH 3.8–4.0. The molecular weights of the enzyme and kininogen were estimated on a G-200 Sephadex column to be 38000–39000 and 115000 respectively. The amount of kinin formed was a function of incubation time and enzyme concentration. The acid protease activity was found localized primarily in the 10000 g supernatant cell fraction. The 500 g cell fraction also exhibited activity.
When plasma comes into contact with a negatively charged surface (e.g., glass, kaolin, collagen) the vasoactive peptide, bradykinin, is generated. Bradykinin can induce two of the cardinal signs of inflammation: Tumor or swelling and dolor or pain. These properties of plasma were first observed and reported by Margolis (1958) and Armstrong et al. (1957), respectively. Bradykinin is cleaved from kininogen by plasma kallikrein. Kallikrein is generated from prekallikrein by activated factor XII (XIIa), the substance in plasma that becomes activated during contact with a negatively charged surface (see Cochrane et al., 1977; Kaplan et al., 1977; Movat, 1978, 1979a) (Fig. 1).
The mechanism of the myostimulating activity of rat tissue kallikrein on rat uterus was re-examined using uterus from kininogen-deficient rats and HOE 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]bradykinin), a specific bradykinin receptor-B2 antagonist. The uterus from kininogen-deficient rats was 50 times less sensitive to rat kallikrein than that from normal rats. HOE 140 (6 to 60 nM) inhibited the contracting effects of bradykinin and of rat kallikrein. Porcine kallikrein had no effect on rat uterus. Bradykinin and rat kallikrein induced a relaxation of rat duodenum. The duodenum from kininogen-deficient rats was 100 times less sensitive to rat kallikrein than the duodenum from normal rats. HOE 140 (0.6 to 3 nM) inhibited the relaxing effects of bradykinin and of kallikrein. Preincubation of rat kallikrein with aprotinin (Trasylol) abolished the effects of kallikrein on smooth muscles. HOE 140 inhibited the amidolytic activity of tissue kallikrein with a Ki value of 220 microM. HOE 140, at micromolar concentrations, suppressed the kininogenase activity of tissue kallikrein. Plasma of deficient rats contained 0.7% of the normal levels of kininogens. After washing the blood vessels with saline, kininogens were present in uterine homogenates but not in duodenal homogenates from both rat strains.(ABSTRACT TRUNCATED AT 250 WORDS)
THE formation and possible function of plasma kinins have attracted considerable interest during recent years. The kinins have been shown to be formed from a fraction within the alpha-2 globulins of the plasma by the action of several enzymes. Kinin-forming enzymes are present in several glandular secretions, such as saliva and sweat. Another type of enzyme, called plasma kallikrein, is present in an inactive form in plasma. The first two plasma kinins identified were bradykinin1 and kallidin2,3. Recently a third, methionyl-lysyl-bradykinin, has been prepared from bovine plasma4. It is not known, however, whether the various kinins are formed from one and the same substrate or if they originate from different substrate fractions within the alpha-2 globulins of the plasma. Nor is it known whether the various enzymes attack one and the same substrate molecule, or if they act on different ones.
DURING investigations on a substrate-plasma for plasmakinin-forming enzymes1, human saliva was used as one source of such enzymes. It has previously been well established that human saliva contains plasma-kinin-forming enzymes2. We found that, when 0.2 ml. saliva was added to 1 ml. substrate-plasma which was free from kininase activity, considerable amounts of plasma-kinin were formed; but it was eventually broken down. This finding seemed to indicate that human saliva contained kininase activity. To investigate if that was really the case, synthetic bradykinin (Sandoz, Ltd.) 500 ng/ml. was added to the saliva and the mixture repeatedly tested for bradykinin activity on a rat uterus suspended in aerogen-ated de Jalons solution. The bradykinin activity disappeared rapidly from the mixture (Fig. 1). The kininase activity was not equally intense in all salivas tested.
1. Correlation between elution volume, V(e), and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (alpha-crystallin) on Sephadex G-200 columns at pH7.5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V(e)-log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V(e)-log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of ;typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including gamma-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10(6). 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of gamma-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.
Aged solutions of tannic acid and of certain o-dihydroxyl compounds similar in structure to its gallic acid moiety accelerated clotting. One of many compounds tested, ellagic acid (4,4′,5,5′,6,6′-hexahydroxydiphenic acid 2,6:2′,6′-dilactone), at concentrations as low as 10-8M, accelerated clotting and appeared to activate Hageman factor in a manner analogous to that of glass surfaces. The chemical nature of the action of ellagic acid was not elucidated but seemed to be related to the presence of o-dihydroxyl groups in its molecule.
Hog pancreas kallikrein does not cause a blood pressure drop in the rat, but lowers the blood pressure in the dog. Since this lack of effect could be due to the inability of hog pancreas kallikrein to act upon rat plasma, the in vitro release of kallidin with the plasma of different species was studied. Human, dog, swine, rat, guinea pig, and ox plasmas were incubated for different periods of time with hog pancreas kallikrein, and the bradykininogen (kallidinogen) consumed and the kallidin released were measured. It was found that hog pancreas kallikrein does not release any kallidin with rat and guinea pig plasma; it releases small amounts with pig plasma and greater amounts with bovine, dog, and human plasma. The bradykininogen consumption showed the same sequence.
The gel-filtration behaviour of proteins related to their molecular weights over a wide range Kallikrein (Padutin) Stuttgart: Ferdinand Enke Verlag Separation of two different substrates for plasma kinin-forming enzymes hind limbs of dogs and rabbits
J C Fasciolo
E K Frey
Kininase activity of human saliva. AMUNDSEN, E. & NUSTAD, K. (1964). 1226-1227. ANDREWs, P. (1965). The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J., 96, 595-606. FAscioLo, J. C. & HALVORSEN, K. (1964). 901-905. FREY, E. K., KRAuT, H. & WERLE, E. (1950). Kallikrein (Padutin). Stuttgart: Ferdinand Enke Verlag. JACOBSEN, S. (1966a). Br. J. Pharmac. Chemother., 26, 403-411. JACOBSEN, S. (1966b). Separation of two different substrates for plasma kinin-forming enzymes. Nature, Lond., 210, 98-99. JACOBSEN, S. (1966c). hind limbs of dogs and rabbits
Some properties of Kallidin, Bradykinin and Wasp Venom Kinin. Polypeptides Which Affect Smooth Muscles and Blood Vessels Ober die Wirkung des Kallikreins auf den isolierten Darm und uber eine neue darm-kontrahierende Substanz
ScHAcHrEm, M. (1960). Some properties of Kallidin, Bradykinin and Wasp Venom Kinin. Polypeptides Which Affect Smooth Muscles and Blood Vessels, p. 237. London: Pergamon Press. WERLE, E. (1937). Ober die Wirkung des Kallikreins auf den isolierten Darm und uber eine neue darm-kontrahierende Substanz. Biochem. Z., 289, 217-233.
Kallikrein (Padutin) Substrates for plasma kinin-forming enzymes in human, dog and rabbit plasmas
E K Frey
FREY, E. K., KRAuT, H. & WERLE, E. (1950). Kallikrein (Padutin). Stuttgart: Ferdinand Enke Verlag. JACOBSEN, S. (1966a). Substrates for plasma kinin-forming enzymes in human, dog and rabbit plasmas. Br. J. Pharmac. Chemother., 26, 403-411.
E K Kraut
FREY, E. K., KRAuT, H. & WERLE, E. (1950). Kallikrein (Padutin). Stuttgart: Ferdinand Enke Verlag.
Some properties of Kallidin, Bradykinin and Wasp Venom Kinin. Polypeptides Which Affect Smooth Muscles and Blood Vessels
ScHAcHrEm, M. (1960). Some properties of Kallidin, Bradykinin and Wasp Venom Kinin. Polypeptides
Which Affect Smooth Muscles and Blood Vessels, p. 237. London: Pergamon Press.