[Primary structure of the intracellular serine proteinase from Bacillus amyloliquefaciens. III. Amino acid sequence of peptides obtained by hydrolysis with a Glu,Asp-specific proteinase. Reconstruction of the entire amino acid sequence of the proteinase]. / Pervichnaia struktura vnutrikletochnoi serinovoi proteinazy Bacillus amyloliquefaciens. III. Aminokislotnaia posledovatel'nost' peptidov, poluchennykh gidrolizom Glu,Asp-spetsifichnoi proteinazoi. Rekonstruktsiia polnoi aminokislotnoi posledovatel'nosti proteinazy.

Glu,Asp-specified protease hydrolysate of intracellular serine proteinase (ISP) was separated by ion-exchange chromatography on a sulphocationite resin followed by HPLC to yield 30 individual peptides. Their sequences, spanning to 243 amino acid residues, were determined by the manual Edman procedure. Four overlapping fragments were reconstructed by comparing their sequences with those of tryptic and chymotryptic peptides. To arrange these fragments in the proteinase polypeptide chain and to reconstruct the enzyme's total sequence, additional peptides were isolated from the tryptic hydrolysate and analysed. Primary structure of ISP, corresponding to 297 amino acid residues, was reconstructed. Its comparison with related serine proteinases revealed the following levels of homology: with Bacillus subtilis intracellular serine proteinase, 88%; with secretory subtilisin BPN' produced by B. amyloliquefaciens, 46%.

[Primary structure of an intracellular serine proteinase from Bacillus amyloliquefaciens. II. Amino acid sequence of peptides in a chymotrypsin hydrolysate]. / Pervichnaia struktura vnutrikletochnoi serinovoi proteinazy Bacillus amyloliquefaciens. II. Aminokislotnaia posledovatel'nost' peptidov khimotripticheskogo gidrolizata.

Chymotrypsin hydrolyzate of the intracellular serine protease was separated by ion-exchange chromatography on a sulphocationite resin followed by HPLC to yield fifty one individual peptides. Their sequences, corresponding in total to 381 amino acid residues, were determined by the manual Edman procedure.

[Primary structure of intracellular serine proteinase from Bacillus amyloliquefaciens. I. Isolation of the enzyme and amino acid sequence of peptides of tryptic hydrolysate]. / Pervichnaia struktura vnutrikletochnoi serinovoi proteinazy Bacillus amyloliquefaciens. I. Vydelenie fermenta i opredelenie aminokislotnoi posledovatel'nosti peptidov tripticheskogo gidrolizata.

Method of isolation of intracellular serine protease was modified. Gramicidin S-sepharose CL-4B with a higher content of the ligand, synthesized through a modified procedure, was used as an affinity sorbent which simplified the purification and led to the pure enzyme with high specific activity and 90% yield. Trypsin hydrolyzate of the protease was separated by ion-exchange chromatography on a sulphocationite resin followed by paper chromatography and paper electrophoresis to yield twenty-five individual peptides. Their complete or partial sequences, corresponding in total to 146 amino acid residues, were determined by the manual Edman procedure.

[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.