Evidence for the promoting role of the intra-uterine kinin release in the development of late hypertonic saline-induced abortion.

Blood plasma kininogen (K), kininases (KS), kallikrein (KK), prekallikrein (PKK), and PGF2a were estimated in the common circulation of pregnant women during late saline-induced abortion and also in retroplacental blood after foetus delivery. The results provide evidence for intra-uterine kinin release from circulating blood K by locally activated KK from the very beginning of abortion. The greatest kinin release coincided with the strongest KS activity decrease at the time of foetus delivery. The pre-abortive KS levels correlated directly with abortus duration. Uterine PG biosynthesis was activated, but appeared to be a secondary process.

PIP: Although human kininogen involvement in hormonal homeostasis during pregnancy and activation of full-term delivery has been documented, the role of intrauterine kinin release in saline-induced late abortion has not been explored. Thus, kinin-related components in the circulation of 53 women with second-trimester (16-27 weeks) hypertonic saline-induced abortions and 20 nonpregnant controls were investigated. Observed were increases in both inactive precursors such as kininogen (K) and prekallikrein (PKK) and the active enzymes kallikrein (KK) and kinases (KS). The prostaglandin (PG) F-2 alpha-KK level in abortion patients did not differ from that in controls. The period from saline instillation to delivery of the fetus (26.5 +or- 2.4 hours) was accompanied only by K and KS changes; these levels decreased simultaneously, reaching their lowest value at the time of fetal expulsion. A 6-fold increase of KK-like activity was detected at fetal expulsion. Thus, the greatest contractile activity of the uterus coincided with the highest concentrations of free kinins and their longest life-time in the systemic circulation. The duration of abortion was directly associated with KS activity level, suggesting that kinins can be used to estimate abortion duration. Tissue KK synthesized in human myometrium and endometrium is considered responsible for local intrauterine kinin release after saline instillation. Finally, a sharp PGF-2 alpha increase (3.6-fold in the systemic circulation and 33-fold in retroplacental blood) was observed at the time of fetal expulsion. The high myometrial contractile activity at the final stage of saline-induced abortion appears to be achieved through the combined effects of locally released kinins and kinin-stimulated prostaglandins.

Kininase of the Latrodectus tredecimguttatus venom: a study of its enzyme substrate specificity.

The specificity of the peptide hydrolyzing action of a highly purified preparation of kininase from Latrodectus Tredecimguttatus venom was studied by the method of TLC on silica gel with the use of various synthetic peptides as substrates. It was shown that the enzyme cleaves the -Pro(7)-Phe(8)-bonds in BK and AI molecules liberating, correspondingly, the C-terminal dipeptide and tripeptide. Exopeptidase specificity was not revealed in the enzyme activity with the use of a number of free and N-substituted tri- and pentapeptides. The results obtained characterize the spider venom kininase as a thiol endopeptidase, which cleaves internal peptide bonds at the proline carboxyl end.

[Activity of tissue and plasma kallikrein and their precursor levels in rabbit tears after alkaline burns of the cornea]. / Aktivnost' tkanevogo i plazmennogo kallikreinov i soderzhanie ikh pred shestvennikov v sleznoi krolikov posle shchelochnogo ozhoga rogovitsy.

Alterations in the activity of tissue and plasma kallikreins and in the levels of their precursors in rabbit lacrimal fluid were studied after alkaline burn of the cornea and treatment with hordox. The enzymatic activity was measured using the fluorogenic substrate Z-Phe-Arg-MCA. The activity of plasma kallikrein was increased in the lacrimal fluid within the first week after burn. Unlike plasma kallikrein, tissue kallikrein showed a higher activity within 14 and 28 days. The maximal increase in the levels of plasma and tissue kallikreins occurred within the first week. Subconjunctival injections of hordox, 0.3 ml (30,000 kIU), daily within 10 days led to a decrease in the activity of either kallikrein within the first week and did not alter tissue kallikrein activity within 14 and 28 days. Hordox did not affect the content of prekallikreins. The activation of tissue kallikrein at days 14-28 appears to occur due to increased proliferation and served as protective and compensatory responses; treatment with the polyvalent inhibitor of proteinases hordox within 8-10 days after burn did not affect this function of tissue kallikrein.

[The status of various indicators of the protease inhibitor system in blood and synovial fluid in rheumatoid arthritis]. / Sostoianie nekotorykh pokazatelei proteazno-ingibitornoi sistemy krovi i sinovial'noi zhidkosti pri revmatoidnom artrite.

Elastase-like activity, alpha 1-inhibitor of proteinases and acid stable antitryptic activity were studied in blood of 26 patients and in synovial fluid of 11 patients with rheumatoid arthritis during acute stage of the disease and after treatment with salase pyridasine and orthophene. The higher values of the elastase-like activity were detected in synovial fluid as compared with that of blood. In blood of these patients, hyperproduction of alpha 1-inhibitor of proteinases and acid stable inhibitors was observed, while deficiency of these substances occurred in synovial fluid. Distinct decrease in the patterns of the blood protease-inhibitory system studied simultaneously with clear positive clinical effect were observed after treatment with salase pyridasine together with orthophene; estimation of these patterns may be used in the evaluation of the therapy.

Inhibition of plasma kallikrein. Kininase and kinin-like activities of preparations from the medicinal leeches.

The medicinal leech salivary gland secretion deprived of hirudin antithrombin activity inhibits amidolytic (substrate S-2302) and kininogenase (substrate kininogen) activities of plasma kallikrein, the main component of the intrinsic mechanism of blood coagulation. It therefore possesses high anticoagulant properties. Kininase (substrate bradykinin) activity of leech saliva and extracts from the medicinal leeches, as well as kinin-like effects of extracts heated at 100 degrees C have been detected. The last one is correlated with the hyperalgetic property of the heated extract. An analgetic effect was observed with the unheated extract but not with leech saliva after intranasal administration to rats.

Components of plasma and tissue kallikrein-kinin system in the urine of patients with latent, nephrotic and hypertonic forms of glomerulonephritis.

Activities of main components of KKS were estimated in the urine of patients with latent, nephrotic and hypertonic forms of chronic glomerulonephritis (ChGN) and compared to those parameters in urine of healthy persons. The data obtained allow to make a conclusion concerning the pathogenetic and the compensatory role of plasma KKS in the nephrotic form of ChGN.

[New dipeptide fluorogenic substrates of human tissue kallikrein]. / Novye dipeptidnye fluorogennye substraty tkanevogo kallikreina cheloveka.

Based on 4-methylcoumarinyl-7-amide (Amc) arginine and a series N-alkyloxycarbonyl derivatives of phenylalanine, eleven Amc-derivatives of the type ROCO-Phe-Arg-Amc (R = alkyl) were synthesized; also were n-C3H7OCO-Leu-Arg-Amc and n-C3H7OCO-D-Phe-Arg-Amc synthesized. The enzymatic hydrolysis of these compounds under the action of tissue and plasma human kallikreins were studied. Tissue kallikrein from human urine hydrolyzed the compounds with R = n-propyl and n-butyl and n-C3H7OCO-Leu-Arg-Amc more readily than the known substrates Z-Phe-Arg-Amc and H-Pro-Phe-Arg-Amc. n-C3H7OCO-D-Phe-Arg-Amc is a weak inhibitor of this enzyme (Ki = 1.5.10(-4) M). Human plasma kallikrein hydrolyzed these novel substrates at a lower rate than Z-Phe-Arg-Amc.

[Activity of tissue and plasma kallikrein and level of their precursors in eye tissue structures and media of healthy rabbits]. / Aktivnost' tkanevogo i plazmennogo kallikreinov i uroven' ikh predshestvennikov v tkanevykh strukturakh i sredakh glaza zdorovogo krolika.

Activity of tissue and blood plasma kallikreins as well as total content of their inactive precursors were studied in rabbit eye structures and media: iris, ciliary body, cornea, vascular striatum, retina, aqueous humor, tear liquid, lacrimal gland by means of fluorimetric procedure using Z-Phe-Arg-MCA as a substrate. Dissimilar capacity of the trypsin soya bean inhibitor and of aprotinin (basic inhibitor of Kunitz type) to inhibit tissue and blood plasma kallikreins enabled to differentiate the enzymatic activity. Lacrimal gland contained the highest activity of tissue kallikrein which amounted to 70% of total Z-Phe-Arg-MCA-hydrolyzing activity of the homogenate. Total Z-Phe-Arg-MCA-amidase activity and activity of individual kallikreins was distinctly lower in all the eye structures and media studied as compared with that of lacrimal gland. Activity of tissue kallikrein was higher than blood serum kallikrein activity in iris, ciliary body, vascular striatum, retina and conjunctiva. The highest content of prekallikreins was found in conjunctiva, aqueous humor, iris and ciliary body. Tissue and blood plasma kallikrein-kinin systems appear to carry out dissimilar functions in eye tissue structures; they are apparently involved in pathogenesis of some eye diseases.

[Activity of elastase-like proteinases and their inhibitors in indigent nearly healthy residents in various biogeochemical conditions of the Chuvash ASSR]. / Aktivnost' élastazopodobnykh proteinaz i ikh ingibitorov u korennykh prakticheski zdorovykh zhitelei v raznykh biogeokhimicheskikh usloviiakh Chuvashskoi ASSR.

A proteinase-inhibitory balance of blood (elastase-like activity, alpha 1-proteinase inhibitor and alpha 2-macroglobulin activities) was studied in practically healthy inhabitants of the Chuvash ASSR two subregions--Sura river basin and Cubninocivil region, which are distinctly dissimilar in all the biogeochemical parameters involving macro- and microtrace compositions. The higher activity of elastase-like proteinases and decreased content of alpha 1-proteinase inhibitor were detected in practically healthy inhabitants of the river Sura basin, where high incidence of myocardial infarction was found, as compared with those of the Cubninocivil people. The similar alterations in the proteinase-inhibitory balance were observed in blood of experimental animals maintained on a diet containing fresh water from these subregions. The data obtained suggest that there exists causative relationship between biogeochemical parameters and development of imbalance in the proteinase-inhibitor system in practically healthy inhabitants of the river Sura basin. This imbalance is considered as a pathogenetic factor responsible for development of atherosclerosis.

[Development of an immunoenzyme method for the quantitative determination of an acid-stable trypsin inhibitor in urine: use for assessing the severity of glomerulonephritis]. / Razrabotka immunofermentnogo metoda kolichestvennogo opredelniia kislotostabil'nogo ingibitora tripsina mochi: primenenie dlia otsenki tiazhesti glomerulonefrita.

Acid-stable trypsin inhibitor was isolated from urine of healthy persons; its homogeneous preparation was obtained using absorption on chitosan, affinity chromatography on chymotrypsin-Sepharose 4B and gel filtration of Sephadex G-100. Two forms of the inhibitor were produced with Mr = 44,000 and 22,000; yield of the trypsin inhibitor was about 70% of its content in urine. Indirect immunoenzymatic assay was developed for the inhibitor estimation; optimal conditions were chosen for sorption of the inhibitor on polystyrene plates as well as for dilution of rabbit anti-inhibitor blood serum and of goat antibodies to rabbit antibodies and horse radish peroxidase conjugates. The procedure developed was sensitive; 10-20 ng of the inhibitor was detected per a sample. Urine of healthy persons and of patients with latent and nephrotic forms of glomerulonephritis was studied using the test system developed. The results of estimation of the trypsin inhibitor were considered in diagnostics and evaluation of severity of glomerulonephritis.

[Purification and some physico-chemical and enzymatic properties of tissue kallikrein from human urine]. / Ochistka i nekotorye fiziko-khimicheskie i énzimaticheskie svoistva tkanevogo kallikreina iz mochi cheloveka.

A procedure for obtaining tissue kallikrein (EC 3.4.21.35) from large specimens of human urea (100 l) has been developed. The isolation procedure included primary extraction of the protein with chitosan (a crustacean chitin deacylated by alkaline treatment), desorption from chitosan with 1 M NH3, affinity chromatography on contrical-Sepharose, ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sephadex G-100. This method permits to obtain tissue kallikrein preparations purified 1080-fold (with respect to AcPheArg-OEt esterase) and 1360-fold (with respect to kininogenase) with 33 and 40% yields, respectively. Tissue kallikrein preparations were homogeneous as could be judged from the results of electrophoresis performed in 12% PAAG in the presence of 0.1% SDS as well as from the presence of one N-terminal amino acid identified as isoleucine. Purified tissue kallikrein had specific activities of 133 mumol/min/mg protein (with respect to AcPheArg-OEt hydrolysis) and 8.8 mumol/min/mg protein (with respect to D-Val-Leu-Arg-pNa hydrolysis) and liberated 462 micrograms equiv. of bradykinin/min/mg protein from heated human blood plasma used as a kininogen source. The protein exhibited the highest stability at pH 8.0-9.0; the pH optimum is at pH 8.0 with AcPheArg-OMe as substrate. The enzyme revealed a high thermostability and was fully inactivated only after 1-hour heating in a boiling water bath. The identity of the urine enzyme to tissue kallikrein could be confirmed by the resistance of the enzyme activity to SIT, high sensitivity to the inhibiting effect of aprotinin (Ki = 0.94 x 10(-10) M) and by an exceedingly low value of the second order inhibition constant for DPP (4.6 M-1 min-1). The fact that this value differs drastically from that for human blood plasma kallikrein (EC 3.4.21.34) which is equal to 360 M-1 min-1 points to marked differences in the structure of the active centers of the both kallikreins as well as to the uniqueness of the tissue kallikrein active center.

[Kininase from the Latrodectus tredecimguttatus venom. Characteristics of the enzyme as a thiol endopeptidase hydrolyzing the -Pro7-Phe8- bond of bradykinin]. / Kininaza iada pauka Latrodectus tredecimguttatus. Kharakteristika fermenta kak tiolovoi éndopeptidazy, rasshchepliaiushchei sviaz' -Pro7-Phe8- v bradikinine.

The nature of the bradykinin (BK)-hydrolyzing (kininase) activity of peptidhydrolase isolated from spider (Latr. tredecimguttatus) venom has been studied. It was found that the BKase activity of the enzyme is fully inhibited by organic mercurials (10(-5)-10(-6) M) as well as by 5,5'-dithiobis(2-nitrobenzoic acid) (10(-7) M); the latter blocks three SH-groups within the enzyme molecule. Serine and metalloproteinase inhibitors have no effect on the kininase activity. Thin-layer chromatography on silicagel revealed that the highly purified enzyme hydrolyzes the -Pro7-Phe8- bond of BK liberating the C-terminal dipeptide, HPhe-ArgOH. Besides, the kininase splits off the C-terminal tripeptide from angiotensin I by hydrolyzing its -Pro7-Phe8-bond. The enzyme does not exhibit any exopeptidase activity with free and N-substituted tri- and pentapeptides. The data obtained suggest that the Latr. tredecimguttatus kininase can be related to thiol endopeptidases hydrolyzing the peptide bonds formed by proline carboxyl.

[A substrate inhibitor analysis of the urinary excretion of tissue and plasma kallikreins in patients with chronic glomerulonephritis]. / Substratno-ingibitornyi analiz mochevoi ékskretsii tkanevogo i plazmennogo kallikreinov u bol'nykh khronicheskim glomerulonefritom.

The usage of substrate inhibitor analysis made it possible to estimate the levels of excretion of plasma proteinases, including plasma kallikrein in the urinary DValLeuArgpNA (S-2266)- and DProPheArgpNA (S-2302)-amidase activity in patients with latent and nephrotic types of chronic glomerulonephritis (CGN). The soya bean trypsin inhibitor, an inhibitor of plasma kallikrein and other plasma proteinases, such as that of the blood coagulative factors XIa and XIIa, and the high selective plasma kallikrein inhibitor DPhePheArgCH2Cl were used as those differentiating kallikreins of tissue and plasma origin. The S-2266 and S-2302-amidase activity of the urine from healthy subjects was shown to be determined by only tissue (renal) kallikrein. The urine from the patients with a latent CGN type displayed the activity of plasma proteinases, but plasma kallikrein made no significant contribution to the urine amidase activity in these patients. With a nephrotic CGN type, great quantities of trypsin-like proteinases were secreted from the plasma through the glomerular filter into the urine, the proportion of plasma kallikrein in the urinary S-2266 and S-2302-amidase activities being approximately 27%. The compensatory and pathogenetic role of plasma kallikrein is discussed if there is lower excretion of tissue (renal) kallikrein in CGN with the nephrotic syndrome.

[Components of kallikrein-kinin system in the urine of patients with glomerulonephritis]. / Komponenty kallikrein-kininovoi sistemy v moche bol'nykh glomerulonefritom.

Activities of main components of the kallikrein-kinin system: tissue (renal) and blood plasma kallikreins, kininases I and II, bradykinin-activating activity as well as free kinins, were estimated in urine of patients with latent and nephrotic forms of glomerulonephritis as compared with those parameters in urine of healthy persons. Content of tissue kallikrein was decreased in urine of patients with nephrotic form of glomerulonephritis as distinct from the disease latent form and healthy persons. At the same time, urine of these patients with nephrotic form contained elevated amount of blood plasma kallikrein and other proteinases of blood plasma origin. Bradykinin-inactivating activity as well as activities of kininase I and II were increased in urine of patients with the disease latent form 2-, 7- and 2.5-fold, respectively, and in urine of patients with nephrotic form--40-, 45- and 40-fold, respectively as compared with normal state. Daily excretion of free kinins with urine of healthy persons constituted 6.6 +/- 0.9 micrograms-eqv of bradykinin (BK), in patients with latent and nephrotic forms of glomerulonephritis--1.7 +/- 0.3 and 2.8 +/- microgram-eqv BK, respectively. Three kinins were found in all the examined persons when urine was collected in acid medium: BK, lysyl-BK and Met-Lys-BK; content of BK was minimal in urine of healthy persons and maximal--in urine of patients with nephrotic form of the disease. Clinical importance of the patterns studied and their role in correction of treatment course of patients with nephrotic form of glomerulonephritis are discussed.

[Photometric method of determination of the amidase activity of proteinases using 4-methylcoumaryl-7-amide substrates]. / Fotometricheskii metod opredeleniia amidaznoi aktivnosti proteinaz s ispol'zovaniem 4-metilkumaril-7-amidnykh substratov.

It was shown that 7-amino-4-methylcoumarin (MC-amine), resulted from the enzymatic hydrolysis of 4-methylcoumaryl-7-amide (MC-amide) peptide substrates, may be estimated not only fluorometrically but also photometrically. A photometric method for estimating activity of tissue kallikrein (EC 3.4.21.35) and urokinase (EC 3.4.21.31) is suggested using Z-Phe-Arg-NHMC and Z-Gly-Gly-Arg-NHMC, respectively, as substrates. Kinetic parameters of the enzymatic hydrolysis, as obtained by photometric and fluorometric detection of the MC-amine formed, were in good agreement. The differential coefficient of molar extinction of the substrates and MC-amine at 360 nm was found to be 10,800 M-1 cm-1.

[Comparative analysis of methods of determination of kallikrein and prekallikrein in the human plasma (review of the literature)]. / Sravnitel'nyi analiz metodov opredeleniia kallikreina i prekallikreina v plazme krovi cheloveka (obzor).

Main procedures developed for estimation of kallikrein activity and of content of its precursor are reviewed; critical comparative analysis of their principles is presented. Specificity, advantages and limitations of these procedures are discussed. General rules of enzymatic kinetics are considered in connection with development and application of these procedures. Some cautions are discussed considering wrong conclusions and recommendations, based on the data obtained by means of the inadequate methods.

[Effective method of simultaneous extraction from the urine and purification of tissue kallikrein and acid-stable trypsin inhibitor]. / Effektivnyi sposob odnovremennogo izvlecheniia iz mochi i ochistki tkanevogo kallikreina i kislotostabil'nogo ingibitora tripsina.

A simple preparative procedure is developed for simultaneous isolation from urine of tissue kallikrein and acid stable trypsin inhibitor. The procedure involved adsorption of these proteins on chitosan at pH 5-6 and the subsequent elution with 1 N NH4OH, which enabled to obtain the enzyme and inhibitor with a yield of 80-90% and to purify 10-fold each of these components. Use of chitosan facilitated and simplified distinctly the large scale isolation from urine of the kallikrein and trypsin inhibitor, required for medicinal and diagnostic purposes.

[A case of hereditary angioneurotic edema provoked by functional insufficiency of C1-inactivator]. / Sluchai nasledstvennogo angionevroticheskkogo oteka, obuslovlennogo funktsional'noi nedostatochnost'iu C1- inaktivatora.

The hemolytic activity of complement, the concentration of C1-inactivator, of C1q and C4 components of complement as well as the activity of contact-activated kallikrein and the functional activity of C1-inactivator were studied in the plasma of a female patient with hereditary angioneurotic edema (HAE), of her mother, sister and normal donors. The hemolytic activity of complement, the concentration of C1-inactivator and C1q in the blood plasma of the persons examined turned out unchanged as compared with the same parameters in donors. Meanwhile in the patient with HAE and her mother with symptoms similar to HAE, the concentration of C4 appeared lower than that in the patient's sister not suffering from the disease and in donors. The activity of contact-activated kallikrein in the plasma of the patient's sister and mother exceeded the activity of the enzyme in donors. The least activity of plasma kallikrein was recorded in the proband. The functional activity of C1-inactivator in the proband constituted 50-60% of the activity of that parameter in her sister and donors. The magnitude of the functional activity of C1-inactivator in the patient's mother approximated its magnitude in the proband. Therefore, the development of HAE in the given case was mainly provoked by functional insufficiency of C1-inactivator. The magnitude of the spontaneous TAME-esterase kallikrein-like activity in the blood plasma of the patient with HAE remained within normal.

[Trypsin inhibitor in the urine of patients with glomerulonephritis]. / Ingibitor tripsina mochi u bol'nykh glomerulonefritom.

Concentrations of urinary trypsin inhibitor (UTI) and acid stable antitryptic activity (AS-ATA) were estimated in morning urine of 50 patients with chronic glomerulonephritis and of 13 healthy persons in order to detect interrelationship between severity of kidney impairment and the content of the inhibitor in urine. In healthy persons concentrations of UTI and AS-ATA were equal to 1.05 +/- 0.15 micrograms/ml and 0.12 +/- 0.04 IU/ml, respectively. Similar values of the substances were detected in patients with latent form of glomerulonephritis and normal kidney function. Statistically distinct (P less than 0.01) increase of both these inhibitors was found in urine of patients with latent form of glomerulonephritis and impaired kidney function (4.77 +/- 1.24 micrograms/ml and 0.39 +/- 0.15 IU/ml, respectively) as well as with nephrotic form (26.17 +/- 7.55 micrograms/ml and 1.37 +/- 0.35 IU/ml, respectively) of glomerulonephritis of both primary type and caused by accompanying systemic diseases. Further increase in concentration of UTI up to 31.74 +/- 7.38 micrograms/ml and activation of AS-ATA in urine was observed in the patients with nephrotic form of glomerulonephritis at the step of chronic kidney insufficiency. The increase in UTI concentration observed did not correlate with the level of leukocyturia. Proteinases of monocytes, mast cells, fibroblasts, involved in inflammation and formation of connective tissue in kidney, but not of enzymes from polymorphonuclear leukocytes, appear to be responsible for formation of UTI out of its precursor inter-alpha-trypsin inhibitor in glomerulonephritis.