Biomedical subjects
Publications and source records attributed to G N Rudenskaia.
The review summarizes characteristics of pathogenic microbial collagenolytic enzymes. Th collagenases usually represent the main factors of virulence; they provide microbe penetration int host tissues and supply microorganisms with nutritients. The study of these enzymes is of great valu for scientific, medical and biochemical purposes.
Trypsin PC from the hepatopancreas of the king crab Paralithodes camtschatica was isolated and purified to apparent homogeneity by ion-exchange chromatography on Aminosilochrom and DEAE-Sephadex and affinity chromatography on arginine-agarose. The yield of the enzyme was 37.7%, and the purification degree was 21. Trypsin PC has a molecular mass of 29 kDa and pI < 2.5. It hydrolysis N-benzoyl-L-arginine p-nitroanilide at the optimum pH of 7.5-8.0 and at the temperature optimum of 55 degrees C (K(m) = 0.05 mM). Trypsin PC retained its activity within the pH range of 5.8-9.0 in the presence of Ca2+. The enzyme was inhibited by the specific inhibitors of serine proteases diisopropyl fluorophoshate and phenylmethylsulfonyl fluoride, by the trypsin inhibitor N-tosyl-L-lysylchloromethylketone, and by the trypsin inhibitors from soybean and potato. Trypsin PC was found to hydrolyze amide bonds formed by carboxylic groups of lysine and arginine in peptide substrates. The N-terminal sequence of this enzyme is IVGGTEVTPG.
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New affine sorbents were synthesized involving tripeptide morpholides H-Ala-Ala-Leu-Mrp and H-D-Ala-Leu-Arg-Mrp as ligands that mimic substrates of subtilisin-like proteases and kallikrein, respectively. These were used for the isolation and purification of several proteases: trypsin, pepsin, alpha-chymotrypsin, thrombin, kallikrein, and termitase and were also efficient in the isolation of proteolytic enzymes from complex mixtures, such as the urine of children suffering from glomerulonephritis, hepatopancreas of Kamchatka crab, and dandelion roots. The ligands are competitive inhibitors of a number of proteases, and therefore, they were supposed to interact with the substrate binding sites in these enzymes.
A homogeneous serine proteinase PC has been isolated from the Camchatka crab (Paralithodes camtschatica) hepatopancreas using affinity chromatography on arginine-Sepharose, protamine tryptic peptide-agarose and ion-exchange chromatography on Mono-Q, with a 68% yield. The enzyme is completely inhibited by diisopropylfluorophosphate, a typical inhibitor for serine proteinases. The molecular mass of the proteinase is 29 kDa, pI is 3.0. The proteinase splits Glp-Phe-Ala-pNA optimally at pH 7.5 and 47-55 degrees C; Km is 0.83 mM, kcat is 67 s-1. The enzyme is stable at pH 4-9. Proteinase PC possesses a broad substrate specificity and splits the peptide bonds formed by the carboxyl group of hydrophobic amino acids, arginine and lysine, in peptides and proteins. The enzyme hydrolyzes fibrin and collagen. Its N-terminal sequence, IVGGQEATP, reveals a 90% homology with analogous sequences of collagenolytic proteinases from other crab species.
Homogeneous carboxypeptidase PC from a hematopancreas of kamchatka crab Paralithodes camtshatica was obtained by means of an affinity chromatography on sorbents containing arginine, protamine hydrolysate, and phenylalanine as ligands with an yield 23% and purification degree 37.4. The isolated enzyme has a molecular mass 34 kDa, as evidenced by an SDS-PAGE; pI 3.1; an optimum pH 6.5, as estimated for hydrolysis of Dnp-Ala-Ala-Arg; pH-stability range 5-8 in the presence of Ca2+; a temperature optimum 55 degrees C; and Km 0.4 mM. The carboxypeptidase is activated by Co2+ and Ca2+ ions and is inhibited by EDTA and o-phenanthroline, and therefore, it is a metallocarboxypeptidase. The enzyme can effectively split off C-terminal residues Phe and Tyr, as well as Arg and Lys. Residues Pro, Glu, and Asp cannot be split off, and they stop the cleaving of a preceding bond. Thus, the carboxypeptidase PC of kamchatka crab has a mixed substrate specificity, which is characteristic of carboxypeptidase from crawfish and of microbial carboxypeptidases T and SG. The new carboxypeptidase has an amino acid composition Asp41Thr24Ser22Glu32Pro15Gly32Ala291/2Cys5Val19Met8Ile14Leu20Ty r18Phe8Lys7His4 Arg8Trp4. The N-terminal sequence of the enzyme demonstrate a 40% homology with the N-terminal sequence of carboxypeptidase from crawfish.
Studies on the affinity chromatography of proteinases as the most efficient approach to their separation are reviewed. The paper contains discussion of the methods used to prepare affinity sorbents by attaching gramicidin and bacitracin to various supports. The two cyclopeptides contain amino acid residues, meeting specificity requirements of proteinases of various classes, these materials thus being affinity sorbents of general type. Rules governing the interaction of proteinases with these sorbents are discussed, along with numerous examples of their chromatography on sorbents of general type as well as on more specific sorbents adapted to the separation of particular types of proteinases. Sorbents containing benzylsuccinic, benzylmalonic and phenylboronic acid residues are considered in details.
Extracellular serine proteinase has been isolated from the cultural medium of Bacillus intermedius 3-19 using CM-cellulose chromatography and affinity chromatography on bacitracin-Sepharose. The specificity of the proteinase with a wide range of natural and synthetic substrates has been investigated. The greatest activity was observed with tripeptides containing C-terminal Leu or Phe. The enzyme was completely inhibited by diisopropylfluorophosphate and partly inhibited by thiol-specific reagents. It is concluded that B. intermedius proteinase is a thiol-dependent serine proteinase pertaining to the subtilisin group. The amino acid composition of the enzyme has been determined. The enzyme contains one to three 1/2 cysteine residues, one of which is supposedly a Cys residue. The N-terminal amino acid sequences of the protein is AQTVPYGIPQIKAPA-.
Different proteins are revealed in cell wall of yeast cells Candida utilis by means of specific proteolysis with subtilisins TV and 72, trypsin and purified collagenase of Clostridium histolyticum. Some of them were characterized by resistance to trypsin and sensitivity to subtilisin TV. In young cells this group is represented essentially by a protein of 33 kD, which appears to be one of the structural proteins, binding fibrillae of carbohydrate. Other proteins proved to be sensitive to both trypsin and subtilisin. Among these proteins a protein with mol. mass 80 kD was revealed; its sensitivity to extremely specific hydrolysis by bacterial collagenase suggests it to contain amino acid sequences characteristic for collagens of higher eukaryotes.
Recent data on serine proteinases belonging to the subtilisin family are reviewed. The following subtilisin subfamilies are discussed: thiol-dependent serine proteinases, intracellular serine proteinases of bacilli, subtilisin-like proteinases from various taxons (archaebacteria, fungi, yeasts, plants, animals).
A homogeneous serine proteinase was isolated from cultural filtrates of the extreme halophilic bacteria Halobacterium mediterranei 1538 using affinity chromatography on bacitracin-Sepharose, ultrafiltration and gel filtration on Sephadex G-75, with a 48% yield and 260-fold purification. The enzyme was completely inactivated by specific inhibitors of serine proteinases, PMSF and DFP, as well as by Hg2+ and PCMB. The enzyme activity was strongly dependent of NaCl concentration, the enzyme being inactivated below 0.75 M NaCl. Inactivation of the enzyme was also seen in the presence of 2-7% organic solvents. The pH optimum for Glp-Ala-Ala-Leu-pNA hydrolysis is 8.0-8.5; Km is 0.14 mM, kcat is 36.9 s-1. The stability optimum lies at pH 5.5-8.0, temperature optimum is at 55 degrees C. The enzyme molecular weight is 41,000 Da; pI is 7.5. The substrate specificity of the enzyme is comparable to that of secretory subtilisins; the extent of protein substrate hydrolysis is similar to that of proteinase K. The N-terminal sequence of Halobacterium mediterranei serine proteinase, Asp-Thr-Ala-Asn-Asp-Pro-Lys-Tyr-Gly-Ser-Gln-Tyr-Ala-Pro-Gln-Lys-Val-Asn- Ala- Asp-, reveals a 50% homology with the aminoterminal sequence of Thermoactinomyces vulgaris serine proteinase. Hence, the serine proteinase secreted by halophilic bacteria may be considered as a structural and functional analog of eubacterial enzymes.
The aminopeptidase was isolated from cell-free extracts of Xanthomonas rubrilineans by protein precipitation by isopropyl ester with subsequent purification by affinity chromatography on CABS-Sepharose, bacitracin-Sepharose, gel filtration through Sephadex G-200 and ultrafiltration, the total yield being 32% with 2200-fold purification. The enzyme was homogeneous during SDS-PAAG electrophoresis. Apart from the broad spectrum of the peptidase activity, aminopeptidase possesses a hydrolase activity towards beta-lactam antibiotics and an esterase activity towards L- and D-amino acids. Besides, this enzyme catalyzes the acetyl transfer reaction during cephalexin synthesis from the D-phenylglycine ester and 7-aminodesacetoxycephalosporanic acid. The maximal enzyme activity during L-Ala-pNA and cephalexin hydrolysis is manifested at pH 6.5. The enzyme is stable at pH 4.0-8.0 and is inhibited by o-phenanthroline, p-chloromercuribenzoate, hydrogen acetate and N-bromosuccinimide. The molecular mass of the enzyme is 270-280 kDa. The enzyme is a tetramer; the molecular mass of each of its four subunits is 70 +/- 2 kDa. The isoelectric point for the enzyme is 6.8. The amino acid composition of the enzyme appears as follows: Asp63, Thr33, Ser32, Glu72, Gly55, 1/2Cys3-4, Val45, Ile24, Leu53, Tyr23, Phe24, Lys23, His16, Arg36, Pro60, Met25, Ala55.
The cultural filtrates of S. thermovulgaris contain a proteinase which is active towards the chromogenic subtilisin substrate, Z-Ala-Ala-Leu-pNa, and azocasein. Pure enzyme preparations were obtained by affinity chromatography on bacitracin-Sepharose with subsequent rechromatography on the same adsorbent. The proteinase was completely inactivated by PMSF and DFP, the specific inhibitors for serine proteinase, by thiol reagents (HgCl2, PCMB) and by the protein inhibitor from S. jantinus. The pH activity optimum for the enzyme is 7.8-8.2, temperature optimum is 55 degrees C. The enzyme is stable at pH 6-9, has a pI of 5.0 and a molecular mass of 32 kDa. When tested against the peptide substrate, the enzyme shows a specificity characteristic for subtilisins. The N-terminal sequence of the enzyme, Tyr-Thr-Pro-Asn-Asp-Pro-Tyr-Phe-Ser-Ser-Arg-Gln-Tyr-Gly, shows a 100% homology with that of terminase, a thiol-dependent serine proteinase. On the basis of the above considerations the enzyme may be related to the subfamily of thiol-dependent serine proteinases.
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Extracellular carboxypeptidase was isolated from culture filtrates of Str. spheroides strain 35, using affinity chromatography on bacitracin-silochrome, bacitracin-Sepharose and CABS-Sepharose. The electrophoretically homogenous enzyme was obtained with a 44% yield and 4160-fold purification. The enzyme-molecular weight is 33,000 Da; pI is 4.7. The amino acid composition of carboxypeptidase is as follows: Asp43, Thr30, Ser35, Glu33, Pro30, Gly47-50, Ala38, 1/2 Cys5-6, Val16, Met2, Ile11, Leu15, Tyr8, Phe10, Lys10, His6, Arg9. The enzyme shows an activity optimum at pH 7.5 is stable at pH 6-8, is completely inhibited with EDTA and can be reactivated by Ca2+. The carboxypeptidase from Str. spheroides strain 35 has a dual substrate specificity, i. e., it splits N-substituted di-, three- and tetrapeptides having both neutral and basic amino acids at the C-ends similar to mammalian carboxypeptidases A and B. The enzyme belongs to the family of metallocarboxypeptidases; its properties are very similar to those of carboxypeptidase S from Str. griseus K-1 and of carboxypeptidase T from Thermoactinomyces sp.
Carboxypeptidase T, an extracellular carboxypeptidase from Thermoactinomyces sp. was isolated and purified by affinity chromatography on bacitracin adsorbents. The enzyme homogeneity was established by SDS electrophoresis (Mr = 38 000) and isoelectrofocusing in PAAG (pI 5.3). Carboxypeptidase T reveals a mixed specificity in comparison with pancreatic carboxypeptidases A and B and cleaves with nearly the same efficiency the peptide bonds formed by the C-terminal residues of basic and neutral hydrophobic amino acids. The enzyme is insensitive to serine and thiol proteinase inhibitors but is completely inhibited by EDTA and o-phenanthroline. The maximal enzyme activity is observed at pH 7-8. With an increase of temperature from 20 to 70 degrees C the enzyme activity is enhanced approximately 10-fold. In the presence of 1 mM Ca2+ the enzyme thermostability is also increased. In terms of some properties, e.g. substrate specificity carboxypeptidase T is similar to metallocarboxypeptidase secreted by Streptomyces griseus. The N-terminal sequence of carboxypeptidase T: Asp-Phe-Pro-Ser-Tyr-Asp-Ser-Gly- Tyr-His-Asn-Tyr-Asn-Glu-Met-Val-Asn-Lys-Ile-Asn-Thr-Val-Ala-Ser-Asn-Tyr- Pro-Asn - Ile-Val-Lys-Thr-Phe-Ser-Ile-Gly-Lys-Val-Tyr-Glu-Gly-Xaa-Gly-Leu- coincides by 21% with that of pancreatic carboxypeptidases A and B. Thus, it may be concluded that these enzymes originate from a common precursor.
Using ion-exchange and affinity chromatography and isoelectrofocusing, eight forms of pepsin with pI 1.6, 1.8, 2.1, 2.3, 2.6, 2.8, 3.2 and 3.6, were isolated from horse gastric juice. The molecular weights, amino acid composition, N-terminal sequence and functional activity of these multiple forms were determined. Partial primary structure of tryptic peptides of pepsin with pI 2.3 was investigated. The analyzed partial sequences of the forms with pI 1.8, 2.1, 2.3, and 2.6 have identical structures which differ from the amino acid sequence of pepsin with pI 3.2 by four substituents. In terms of their functional activity, horse pepsins differ only insignificantly. Presumably, the pepsins under study (at least the forms with pI 1.8, 2.1, 2.3, 2.6 and 3.2) arose comparatively recently as a result of duplication of the common precursor gene and exist at an early stage of structural and functional divergence. As far as their primary structure and functional properties are concerned, these pepsins are more related to pepsin A than to other isoenzymes of gastric aspartyl proteinases of mammalia, e. g., gastricsin or chymosin.