Evidence for the presence of dipeptidyl carboxypeptidase and its inhibitors in inflammatory synovial fluids.

Evidence is reported for the presence of dipeptidyl carboxypeptidase (peptidyldipeptide hydrolase, EC 3.4.15.1) and of inhibitor(s) of this enzyme in synovial fluids from patients with rheumatoid arthritis and gout.

Effect of kininogen on the activity of the kallikreinogen-kallikrein system of human blood serum.

In experiments with kallikreinogen, kallikrein and kininogen preparations from human blood serum partially purified on QAE-Sephadex A-50, CM-Sephadex and Sephadex G-200 it was established that N-benzoyl-L-arginine ethyl ester (BAEE)-esterase activity of kallikrein and the process of autoactivation of kallikreinogen are inhibited by kininogen, especially by its high-molecular form.

Isolation of a cationic fragment of high molecular mass kininogen and effect of this fragment on human plasma kallikrein activity.

The effect of a cationic fragment of high molecular mass (HMM) kininogen on kallikrein activity and the autocatalytic process of prekallikrein-to-kallikrein conversion were studied. Prekallikrein and kallikrein were obtained by ion exchange and affinity chromatography; the purification of HMM kininogen was achieved using QAE- and DEAE-Sephadex, and separation of the cationic fragment was carried out by gel filtration. It was shown that the fragment of HMM kininogen (mol. mass. 7000, high lysine content) suppressed the activity of a labile form of kallikrein and prevented activation of prekallikrein. A model of the prekallikrein-kallikrein system is proposed. The model is based on the recently established fact of occurrence of two kallikreins and two kallikrein precursors. It involves formation from kininogen, besides kinins, of a kallikrein inhibitor.

Preparation and some properties of highly purified human serum kallikrein.

A 3000--6000-fold purified kallikrein was obtained from human serum in 10--25% yield by chromatography on QAE-Sephadex A-50, Molselect CM-50 and on soybean trypsin inhibitor (SBTI)-AH-Sepharose 4-B. The enzyme had a specific activity of 14--23 U, as measured by BAEE hydrolysis. Some properties of highly purified kallikrein are described.

Preparation and some properties of human serum kallikrein immobilized on ARM-Sepharose.

A highly purified human serum kallikrein immobilized on CH-Sepharose 4-B was obtained. KM values for N-benzoyl-L-arginine ethyl ester and N-tosyl-L-arginine methyl ester hydrolysis of this preparation were 1.10 x 10(-3) M and 3.6 x 10(-4) M, respectively; pH optimum of hydrolysis of these esters were found to be 8.2 and 8.5, respectively. The immobilized kallikrein possessed kininogenase activity and was capable of activating prekallikrein.

[Endocrine function of the heart. Structure and biological properties of peptides secreted by the heart atrium]. / Endokrinnaia funktsiia serdtsa. Struktura i biologicheskie svoistva peptidov, sekretiruemykh predserdiiami.

Apart from the generally known functions, the heart has also an endocrine function. Atrial cardiocytes, being typical secretory cells, release peptide hormones into the blood stream: atrial natriuretic peptide containing 28 amino acids and cardiodilatin. The structure of atrial peptides was determined. It was shown that both peptides were derived from their common precursor, a protein containing 151 amino acids. The presence of specific receptors is demonstrated on plasmatic membranes of cells of kidney epithelium, arterial smooth muscle, arterial endothelium, kidney cortex and hypophysis. The interaction of atrial peptides with these receptors activates the guanylate cyclase system. The biological action of atrial peptides manifests itself in the quick, massive and instantaneous increase of diuresis and electrolyte excretion, elevated clearance of creatinine, decrease of kidney vascular resistance, intensification of glomerular filtration, inhibition of stimulated secretion of aldosterone, relaxation of blood vessels, elimination of arterial and intestinal spasm induced by various endogenous and exogenous vasoconstrictors and in correction of kidney hypertension. Various radioimmunoassays for the presence of atrial peptides in human plasma were developed; it was shown that in patients with congestive heart failure the content of atrial peptides is increased.

Presence of a proteinase in polyribosomes of rat liver.

The presence of a proteinase in the composition of the ribosomal proteins of rat liver is demonstrated. The enzyme possesses optimal activity in the zone of pH 7.0-7.2. Soybean trypsin inhibitor and 1-chloro-4-phenyl-3-tosylamido-2-butanone inhibit the enzyme by 50-60%.

[Synthesis and analysis of conformationally restricted analogs of peptide inhibitors of the angiotensin-converting enzyme]. / Sintez i issledovanie konformatsionno ogranichennykh analogov peptidnykh ingibitorov angiotenzinprevrashchaiushchego fermenta.

To investigate conformations of peptide inhibitors of the angiotensin-converting enzyme in the enzyme-inhibitor complex, the synthesis, studies of inhibitory activity, and conformational calculations of analogues of bradykinin-potentiating peptides with N-methylalanine or D-alanine in place of L-proline or L-alanine residues have been carried out. All the analogues showed a sharp decrease of inhibitory activity in comparison with the natural peptides, that might be considered as an indirect confirmation of the earlier proposed "conformation of inhibition" of the above-mentioned peptides.

[Isolation and purification of biopolymers using an affinity chromatography method. IX. Preparation, properties and use of affinity adsorbents with immobilized polypeptide fragments of collagen]. / Vydelenie i ochistka biopolimerov metodom affinnoi khromatografii. IX. Poluchenie, svoistva i primenenie affinnykh adsorbentov s immobilizovannymi polipeptidnymi fragmentami kollagena.

Synthesis and properties of new affinity adsorbents with immobilized polypeptide fragments of collagen molecule (alpha-chains, beta-components, cyanogen bromide peptides) were described. Adsorbents with alpha-chains and alpha 1CB7-peptide had fibronectin binding capacity 1.5-2.0 times higher than commercial gelatin-Sepharose. Commercial production of highly purified fibronectin from human plasma using affinity chromatography on immobilized individual alpha-chains of collagen was developed.

[Changes in dipeptidyl carboxypeptidase activity during hypertension and its complications]. / Izmenenie aktivnosti dipeptidil-karboksipeptidazy pri gipertonicheskoi bolezni i ee oslozhneniiakh.

Using the fluorimetric method, the activity of dipeptidyl-carboxypeptidase (DCP) was studied in 85 patients suffering from essential hypertension. It was ascertained that the DCP activity in the blood serum of such patients was significantly higher than in normal subjects. The highest activity of the enzyme was observed in cases where essential hypertension was aggravated by chronic heart failure or an acute impairment of the cerebral circulation. Inflammatory processes in hypertensive patients were associated with a considerable fall in the serum enzyme activity.

[The peptidase activity of a cellular extract of the causative agent of plague]. / Peptidaznaia aktivnost' v ékstrakte kletok vozbuditelia chumy.

Peptidase activity of Yersinia pestis cell extract was studied by the hydrolysis of synthetic and some natural substrates, as well as the hydrolysis of fluorogenic aminopeptidase substrates. All peptides and fluorogenic substrates under test were split by Y. pestis cell extract, the splitting of alanylaminopeptidase substrate being linked with at least two enzymes of this cell extract.

[A new approach to the problem of cardio-vascular regulation: the endocrine function of the heart (review of the literature)]. / Novoe v probleme serdechno-sosudistoi reguliatsii: éndokrinnaia funktsiia serdtsa (obzor).

Besides generally known functions, the heart has also an endocrine function. Atrial cardiocytes, being typical secretory cells, released peptide hormones into the blood stream: atrial natriuretic peptide containing 28 amino acids and cardiodiolatin. The structure of atrial peptides was determined. It was shown that both derived from their common precursor, a protein containing 151 amino acid. The presence of specific receptors was demonstrated on plasmatic membranes of cells of kidney epithelium, arterial smooth muscle, arterial endothelium, kidney cortex and hypophysis. The interaction of atrial peptides with these receptors activated the guanylate cyclase system. The biological action of atrial peptides manifested itself in the quick, massive and instantaneous increase of diuresis and electrolyte excretion, elevation of clearance of creatinine, decrease of kidney vascular resistance, intensification of glomerular filtration, inhibition of stimulated secretion of aldosterone, relaxation of blood vessels, elimination of arterial and intestinal spasm induced by various endogenous and exogenous vasoconstrictors and correction of kidney hypertension. Various radioimmunoassays for detection of atrial peptides in human blood plasma were developed; it was shown that in patients with congestive failure the atrial peptide content was increased.

[Chromatographic method of determining the activity of the collagenase-like enzyme of the adenohypophysis and several findings concerning the specificity of its action]. / Khromatograficheskii metod opredeleniia aktivnosti kollagenazopodobnogo fermenta adenogipofiza i nekotorye dannye o spetsifichnosti ego deistviia.

A chromatographic method is developed for quantitative estimation of the collagenase-like enzyme (CLE) activity in extract of adenohypophysis and in preparations obtained during various steps of the enzyme isolation. The enzymatic hydrolysis of Cbz-Gly-Pro-Ala-Gly-Pro-Gly-OCH3 in presence of 2-mercaptoethanol at pH 8.0 was used as a pattern. The products formed were separated by chromatography on the paper; then they were stained with ninhydrin and converted into cupric complexes during extraction with ethanol; the optic density was measured at 510 nm. The optimal conditions for the enzymatic reaction were established. The method enabled to estimate the CLE activity in presence of prolyl carboxypeptidase. The specific effect of the CLE purified preparation on various synthetic peptides is discussed.

[Characteristics of cysteine peptidohydrolases from the human kidney cortex]. / Kharakteristika tsisteinovykh peptidgidrolaz iz korkovogo sloia pochek cheloveka.

Spectrum of cysteine peptide hydrolases was studied in human kidney cortex using a variety of protein and synthetic substrates after the preparation was fractionated on Sephadex G-100 following the precipitation with ammonium sulfate 40-70%. Activities of dipeptidyl aminopeptidase I, lysosomal carboxypeptidases A and B, cathepsins B and H, as well as the activity of Ca+2-dependent neutral proteinase were detected. A thiol-dependent proteolytic activity, which was apparently stimulated by cathepsin T- and -M like enzymes, was also observed. Besides, the proteinase with molecular mass of 50-60 KDa and high hemoglobin and casein splitting activity was found. This activity cannot be attributed to any of the proteinases so far known. Isoelectrofocusing technique showed that two forms of cathepsin B occurred in human kidney with isoelectric points at pH 5.32 and pH 5.65; two forms of cathepsin H were also detected exhibiting isoelectric points at pH 6.03 and pH 6.7.

[The role of lysosomal proteinases in tissue destruction]. / Rol' lizosomnykh proteinaz v destruktsii tkani.

Action of lysosomal and other proteolytic enzymes on components of the intracellular matrix--collagen, proteoglycans and fibronectin are reviewed. Various types of extra- and intracellular degradation of the intracellular matrix and interrelationship of these types are discussed. Regulatory functions of lysosomal proteinases and their role in activation of latent collagenase and in inactivation of alpha 1-proteinase inhibitor are considered. Some physiological and pathological processes involving local destruction of tissues are discussed.

[Properties and action specificity of carboxycathepsin (peptidyl dipepsidase) from bovine kidneys]. / Svoistva i spetsifichnost' deistviia karboksikatepsina (petidil-dipeptidazy) pochek byka

Carboxycathepsin from bovine kidney split the dipeptide fragments from the C-terminal part of peptides of different structure. Peptides containing the proline residue at the second position from the C-terminal amino acid residue and also peptides with substituted terminal alpha-carboxyl group were not hydrolyzed by carboxycathepsin. The enzyme was activated by Cl, Zn2+, Co2+ and Mn2+. The substances which formed the chelate complexes with ions of two-valent metals and also heavy metal ions, inhibited the enzymatic activity. Diisopropyl fluorophosphate did not inhibit carboxycathepsin. The homogeneous preparation of carboxycathepsin converted angiotensin 1 into angiotensin 11 and hydrolyzed bradikinine, splitting off C-terminal dipeptides consequentially.

[Isolation and properties of 3 Clostridium histolyticum collagenases]. / Vydelenie i svoistva trekh kollagenaz Clostridium histolyticum.

The collagenases (I, II and III) have been obtained in a highly purified state from fresh cultural medium of Clostridium histolyticum. The collagenases were similar in their properties to clostridiopeptidase A. The three enzymes differed in their molecular weights, isoelectric points and in some chemical properties. Collagenase II exhibited the most potent hydrolytic activity. Its collagenolytic activity was two-fold higher and the peptidase activity was twenty-fold higher as compared with that of collagenase I. All the three enzymes were inactive towards azocasein and were inhibited by EDTA and cysteine.

[Immunochemical studies of human cathepsin D]. / Immunokhimicheskie issledovaniia katepsinov D cheloveka.

Rabbit antiserum against human liver cathepsin D was raised. The antiserum did not cross-react with cathepsins D from tissues of other species (bovine liver, bovine spleen, chicken liver). The study of cathepsins D isolated from human pathologically altered tissues showed that cathepsins from kidney malignant tumor and from myeloleucosis-induced spleen tumor were immunologically identical to the enzyme from normal liver. Cathepsins from liposarcoma and uterine myoma were characterized by partial identity with the enzyme from normal liver. Cathepsins D isolated from various human livers exhibited individual quantitative differences in antigenic properties, a fact to be taken into account in development of an immunochemical method for identification of cathepsin D. The low immunogenicity of human cathepsin D for rabbits and inadequate suitability of these animals for raising appropriate antisera was also considered.

[Fluorometric method of determining prolylendopeptidase activity in human erythrocytes in normal and pathologic conditions]. / Fluorometricheskii metod opredeleniia aktivnosti proliléndopeptidaz v éritrotsitakh liudei v norme i pri patologicheskikh sostoianiiakh.

A highly sensitive fluorometric method for determination of prolylendopeptidase (PE) activity in human erythrocyte hemolysates in the presence of hemoglobin has been developed. The method is based on measurement of fluorescence of 4-methyl-7-aminocoumarine released in the course of enzymatic reaction from the substrate Z-glycyl-proline-4-methylcoumarine-7-amide. A correlation was introduced for the quenching of fluorescence by hemoglobin. The method is suitable for the determination of PE activity in human erythrocyte hemolysates in various pathological states. The dependence of PE activity on the incubation time, protein and substrate concentrations were studied using the 1,200-fold purified preparations of prolylendopeptidase II. The values of PE activity in erythrocyte hemolysates of healthy donors and in those of patients with odontogenic phlegmons of maxillary-facial area were virtually identical. PE activity in erythrocyte hemolysates of stored blood was 5 times lower than that in the cell hydrolysates of fresh blood. The PE activity was not observed in blood serum of fresh and stored blood of healthy persons and of patients with acute inflammatory processes of maxillary-facial area, as well as in blood serum of patients with hepatitis and glomerulopephritis.

[Determination of carboxycathepsin (peptidyl dipeptidase) activity in human blood serum]. / Opredelenie aktivnosti karboksikatepsina (peptidil-dipeptidazy) v syvorotke krovi cheloveka

Based on fluorometric determination of a dipeptide histidil-leucine, which is splitted off from a synthetic substrate (Cbz-Phe-His-Leu) by carboxycathepsin (carboxydipeptidyl peptide hydrolase), a method was developed for estimation of the enzymatic activity in human blood serum. The method is characterized by simplicity and high sensitivity; Use of small amount of blood serum (about 0.03 ml) was possible. In normal human blood serum the carboxycathepsin activity varied from 7.5 to 18 nmoles of the dipeptide, liberated per mg of protein per hr.