[Renaturation of bacterial metalloproteinases]. / Renaturatsiia bakterial'nykh metalloproteinaz.

After denaturation by phenol or acid ethanol bacterial metalloproteinases secreted by Bacillus thermoproteolyticus and Bacillus megaterium cells can be renatured by dissolution in 99.7% formic acid with subsequent dilution of the solution and its neutralization with an appropriate amount of an alkali. Renaturation is optimal at pH 9.0 in the presence of 30% glycerol as stabilizer, Ca2+ and Zn2+ ions needed for the formation of a native structure and reconstitution of the enzyme catalytic center. The active enzyme yield is 60-80%. Reactivated metalloproteinases retain their enzymatic properties, amino acid composition and molecular mass. Under these conditions autolysis of metalloproteinases does not significantly influence their renaturation.

[Isolation and properties of intracellular peptidase from Brevibacterium]. / Vydelenie i svoistva vnutrikletochnoi peptidazy Brevibacterium.

The intracellular peptidase of Brevibacterium E531, a lysine-producing bacterial species, was purified 6500-fold by chromatography on DEAE-cellulose and the affinity adsorbent H-Thr(But)-Phe-Pro-hexamethylene-diamine-Sepharose 4B and by gel filtration on Sephadex G-200. The enzyme displayed the maximum activity towards proline p-nitroanilide at pH 7.7-7.9 and readily split glycine, alanine and proline from di-, tri- and tetrapeptides but did not practically hydrolyze oligopeptides of a greater chain length. The enzyme was not inhibited by complexons (EDTA, 8-oxiquinoline and 1.10-phenanthroline). The peptidase was not activated by divalent metal ions and was inhibited by Zn2+; Cd2+, Hg2+ and Cu2+. Data brom gel filtration on Sephadex G-200 suggest that the molecular mass of the enzyme is no less than 250 kDa. In the presence of sodium dodecyl sulfate the molecular mass of the enzyme is 43 kDa, which is suggestive of the presence of a quaternary structure. One peculiarity of the enzyme is its activation by alkaline metal halogenides and sodium nitrate which reaches a maximum at the 0.05-0.1 M concentration of the salts.

[Pepsinogen activation by microbial proteinases]. / Aktivatsiia pepsinogena proteinazami mikroorganizmov.

Serine proteinase and metalloproteinase of Asp. oryzae, extracellular metalloproteinase of L. pneumophila and chymotrypsin-like proteinase of S. rutgersensis can hydrolyze pepsinogen by converting it into pepsin (pH 5.0, 37 degrees C). The localization of the site of hydrolysis depends on the nature of the enzyme: serine proteinase from Asp. oryzae induces the synthesis of a mixture of 60% pepsin, 25% leucyl-pepsin and 15% alanyl-leucyl-pepsin; metalloproteinase of Asp. oryzae converts pepsinogen only into leucyl-pepsin, while metalloproteinase of L. pneumophila yields a mixture of 33% pepsin, 53% leucyl-pepsin and 14% alanyl-leucyl-pepsin. Thus, the region of the activating pepsinogen peptide--Ala 42P-Ile 1 bond--seems to the most probable site for hydrolysis by exogenous proteinases. This site contains a Leu 44P-Ile 1 bond which is subjected to intermolecular hydrolysis during autocatalytic activation of pepsinogen. The experimental results emphasize the importance of the intermolecular pathway of pepsinogen activation.

[Isolation and properties of leucine aminopeptidase from Aspergillus oryzae]. / Vydelenie i svoistva leitsinaminopeptidazy iz Aspergillus oryzae.

Homogenious leucine aminopeptidase is purified from "oryzine"--mixture of enzymes produced by surface culture of Asperigillus oryzae using treatment with activated characoal, followed by DEAE-cellulose and hydroxylapatite chromatographies, Biogel P-100 gel-filtration and polyacrylamide-gel electrophoresis. The enzyme has pH optimum 9.0 and the molecular weight 37500 as estimated by gil-filtration through Sephadex G-100 (superfine) and SDS-polyacrylamide gel electrophoresis. Leucine aminopeptidase from Asp. oryzae has a broad substrate specificity, therefore, cleaving with the highest rate the peptides carrying N-terminal leucine. The enzyme is completely inhibited with EDTA and beta-mercaptoethanol, and it is a metalloenzyme.

[Biospecific chromatography of Bacillus subtilis metalloendoproteinase and detection of multiple forms of the enzyme]. / Biosletsificheskaia khromatografiia metalloendoproteinazy Bacillus subtilis i obnaruzhenie mnozhestvennykh form fermenta

Metalloendoproteinase was isolated from protosubtilin, i.e. a mixture of enzymes produced by Bacillus subtilis, during adsorption on activated carbon and a subsequent biospecific chromatography on DNP--hexamethylene diamine--Sepharose 4B and gramicidin S--Sepharose 4B. The molecular weight of the enzyme estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was found to be 36.000. The preparation isolated contained four molecular forms of the enzyme, each splitting DNP-Gly-Gly-Val-Arg. It is shown that thermolysine, purified by biospecific chromatography, consists of three molecular forms. The amino acid composition of metalloendoproteinase was established.

[Isolation and properties of the acid carboxypeptidase of Aspergillus oryzae]. / Vydelenie i svoistva kisloi karboksipeptidazy iz Aspergillus oryzae

Caboxypeptidase with pH optimum 4--5 for peptide substrates is isolated from "oryzine", the enzyme mixture produced by Aspergillus oryzae, by means of successive salt fractionation, Sephadex G-75 gel filtration, chromatography on amberlite IRC-50, hydroxylapatite, DEAE-cellulose and polyacrylamide gel electrophoresis. Its molecular weight, as determined by means of polyacrylamide gel electrophoresis in the presence of sodium dolecylsulphate, was found to be 37 000. The enzyme has a broad substrate specificity and does not possess dipeptidase and esterase activities. Acid carboxypeptidase from Asp. oryzae is not a metalloenzyme, it is inactivated by specific inhibitors of serine proteases and by compounds blocking SH-groups. The enzyme is suggested to contain functionally important serine and cysteine residues and to be acid carboxipeptidase.