[Serine proteinase from the higher basidiomycetes of Coprinus genus]. / Serinovaia proteinaza iz vysshego bazidiomitseta roda Coprinus.

A thiol-dependent serine proteinase has been isolated for the first time from a higher basidiomycete Coprinus 7N culture filtrate by affinity chromatography on bacitracin-Sepharose combined with ion-exchange chromatography on DEAE-Sepharose. This procedure resulted in a homogeneous enzyme with 32-fold purification and 55% yield. The enzyme has a molecular mass of 33,000 Da and pI of 8.5; its amino acid composition appears as follows: Lys7, His7, Arg10, Asx29, Thr24, Ser30, Glx19, Pro13, Gly39, Ala40, Cys2-3, Val23, Met1-2, Ile14, Leu13, Tyr6, Phe7. The enzyme shows the optimal activity towards Z-Ala-Ala-Leu-pNA at 8.5 and is stable at pH 6-9. The temperature optimum of the enzyme activity lies at 37 degrees C. The proteinase is completely inactivated by the specific inhibitors of serine proteinases, diisopropylfluorophosphate and phenylmethylsulfonylfluoride, as well as by the SH-group reagent, p-chloromercuribenzoate. The Coprinus 7N proteinase hydrolyzes, azocasein, azoalbumin, hemoglobin, fibrin and synthetic chromogenic peptide substrates, e. g., Z-Ala-Ala-Leu-pNA, Z-Gly-gly-Leu-pNA. Some properties of the Coprinus 7N proteinase are very similar to those of thiol-dependent serine proteinases from bacilli, actinomycetes, fungi and plants which form a subfamily of thiol-dependent serine proteinases within the family of subtilisins.

[Isolation of proteinase I from E. coli cells using affinty chromatography on bacitracin-Sepharose]. / Izuchenie proteinaz E. coli. Vydelenie proteinazy I s pomoshch'iu affinnoi khromatografii na batsitratsin-sefaroze.

A serine proteinase (proteinase I) was isolated in a homogeneous state from E. coli K12 cells, using bacitracin-Sepharose 4B affinity chromatography. The enzyme effectively cleaved N alpha-acetyl-L-phenylalanine beta-naphthyl ester. The proteinase was inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, but was resistant to EDTA and natural trypsin or subtilisin protein inhibitors. The enzyme did not cleave trypsin and subtilisin synthetic substrates, possessing a narrow substrate specificity. The amino acid composition of the enzyme was determined. The enzyme molecular weight was found to be about 20 000.

[Immunologic comparison of intracellular and extracellular serine proteases from Bacillus amyloliquefaciens and some other proteases]. / Immunologicheskoe sravnenie vnutrikletochnoi i vnekletochnoi serinovykh proteinaz Bacillus amyloliquefaciens i nekotorykh drugikh serinovykh proteinaz.

Antibodies against intracellular serine protease and extracellular subtilisin BPN' were raised in rabbits. Using these antibodies and antisera against subtilisin Carlsberg and thermitase (serine protease from Thermoactinomyces vulgaris), it was shown that the proteases of the subtilisin family possess a pronounced immunological variability. Immunological studies demonstrated that the vegetative and sporulating B. amyloliquefaciens cells contain no long-lived protein precursor of intracellular serine protease and that the drastic increase of the enzyme activity during the first hours of the sporulating period is presumably due to its de novo synthesis. The specific protein inhibitor of intracellular serine protease partially purified from B. amyloliquefaciens sporulating cells did not prevent the enzyme interaction with its specific antibodies.

[Metalloproteinase from Bacillus subtilis: - "intracellular" and extracellular enzymes]. / Metalloproteinaza iz Bacillus subtilis: vnekletochnyi i vnutrikletochnyi fermenty.

"Intracellular" metalloproteinase was purified to homogeneity from Bacillus subtilis 103 crude cell extract, using affinity chromatography on bacitracin-Sepharose 4B. The degree of purification and the yield of the enzyme were about 260-fold and 3%, respectively. In its physico-chemical properties and the amino acid composition the enzyme is very similar, if not identical, to the extracellular metalloproteinase isolated from the culture filtrate of the same strain. Extracellular metalloproteinase-deficient mutant strain Bacillus subtilis SMY-512 does not produce the "intracellular" enzyme either. THe activity of "intracellular" metalloproteinase in the periplasmic space of the cells is about 70% of that in the cytoplasm, thus being indicative of a rather regular distribution of the enzyme throughout the cell compartment.