[Analysis of the structure of Bacillus brevis neutral proteinase and its biosynthesis in Bacillus subtilis cells]. / Analiz struktury neitral'noi proteinazy Bacillus brevis i ee biosintez v kletkakh Bacillus subtilis.

Amino acid sequence of neutral metalloprotease from Bac. brevis has been compared with that of Bac. amyloloquefaciens, Bac. cereus, Bac. subtilis, Bac. stearothermophilis, Bac. thermoproteolyticus (thermolysine). A sequence region from N-40 to N-1 with a significant degree of homology allowed to predict the processing site of the propart of Bac. brevis enzyme. The sequence comparison allows to put Bac. brevis enzyme within the evolutionary branch of enzymes, which includes thermolysin and proteases of Bac. cereus and Bac. stearothermophilus. Using automated Edman degradation the N-terminal sequence of Bac. brevis protease has been determined. It does not differ from the sequence predicted from the nucleotide sequence of the gene. It was shown that, when Bac. brevis gene coding for thermostable protease is expressed on a plasmid vector in Bac. subtilis cells at 37 degrees C, enzyme forms possessing low activity are secreted. The enzyme may be significantly activated without an additional cleavage or processing and the activation includes numerous conformation transition states of the protein molecule.

[The effect of synthetic proteinase inhibitors of the level of human recombinant proinsulin production, secreted by a genetically engineered strain of Bacillus subtilis]. / Vliianie sinteticheskikh ingibitorov proteinaz na uroven' produktsii rekombinantnogo proinsulina cheloveka, sekretiruemogo genno- inzhenernym shtammom Bacillus subtilis.

Influence of O,O,-diethyl-1-(N-alpha-hydrohexafluoroisobutyryl)amino-1- methylpropylphosphonate and O,O-diisobutyl-1-[2-(ethoxycarbonyl)aminoperfluoroprop-2-yl] -1- methylpropylphosphonate on the level of production of human proinsulin secreted by a genetically engineered culture Bacillus subtilis AJ 73 (pBINS1.0.) has been studied. The above phosphonates, being non-toxic for microorganisms, reduced degradation of proinsulin by serine proteinases.

[Interferon-specific cellular receptors in cultured human cells, differing in sensitivity to alpha-interferon]. / Interferon-spetsificheskie kletochnye retseptory v kul'turakh chelovecheskikh kletok, razlichaiushchikhsiia po chuvstvitel'nosti k alpha-interferonu.

The interferon-specific cellular receptors in human cells cultures differing in sensitivity to alpha-interferon have been studied. The J-41 cells resistant to alpha-interferon are practically devoid of receptors highly specific to alpha-interferon. The coefficient of equilibrium and the number of receptors analyzed after Scatchard for J-96 culture of cells are 15.6 x 10(11) M-1 and 210 +/- 90, respectively. Evidently, resistance of J-41 cells to alpha-interferon is connected with the absence of interferon receptors and, as a consequence, inability to interferon-receptor interaction.

[Mutations in Escherichia coli pmp and htpR genes stabilize the products of foreign gene expression]. / Mutatsii v genakh pmp i htpR Escherichia coli stabiliziruiut produkty ékspressii chuzherodnykh genov.

The half lives of mRNA for Escherichia coli chloramphenicol-acetyltransferase, Bacillus amyloliquefaciens alpha-amylase and human leucocyte interferon were measured in E. coli cells by molecular RNA.DNA hybridization. The effect of mutation in pnp gene, coding polynucleotide phosphorylase, on the stability of these mRNA was studied. The half life of interferon mRNA increases from 25 to 90 s in the pnp mutant, resulting in an increase of interferon accumulation. The stability of interferon in E. coli cells depends on the htpR gene, controlling the heat shock response. The yields of leucocyte interferons alpha-2, alpha I-1 and fibroblast interferon beta increase ten times in htpR mutants. Thus, by using pnp and htpR mutants it is possible to enhance considerably the eukaryotic gene expression in bacterial cells.

[Analysis of oligomeric forms of recombinant human leukocyte interferons]. / Analiz oligomernykh form genno-inzhenernykh chelovecheskikh leikotsitarnykh interferonov.

Using SDS-PAAG electrophoresis, gel-permeation HPLC and immunoblotting, it was demonstrated that homogeneous preparations of human leukocyte interferons (alpha-INF)-A, -N and -I1 obtained from the biomass of the corresponding producer strains (Pseudomonas sp.) contained several oligomeric forms produced by way of S-S intermolecular cross-linkage and making up to 10-15%, 4-7% and 2-5% of the total monomeric form content in the protein preparations. Immunologic testing with the use of MAB NK-2 and [125I]NK-2 showed that the oligomeric forms of alpha-INF-A, -N and -I1 were present in the protein preparations at all purification stages and seemed to be formed at early steps of interferon synthesis in the cell. The effects of limited proteolysis as well as of acid, alkaline and thermodenaturation on the aggregation and oligomerization of alpha-INF-A were studied. SDS-PAAG electrophoresis performed in the absence of the reducing agents showed that upon denaturation of 10% TCA, the amount of the oligomeric forms in the preparations of homogeneous and especially partly proteolytic INF was significantly increased. The causes and the putative mechanisms of aggregation and oligomerization of INF are discussed.

[Molecular organization and pigment composition of R-phycoerythrin from the red alga Callithamnion rubosum]. / Molekuliarnaia organizatsiia i pigmentnyi sostav R-fikoéritrina krasnoi vodorosli Callithamnion rubosum.

p3phycoerythrin is the major phycobiliprotein of Rhodophyta and endows these algae with the characteristic color. R-phycoerythrin purified from red alga Calithamnion rubosom is composed of four dissimilar polypeptide subunits, alpha, beta, gamma, and delta. In calibrated SDS gel electrophoresis their molecular weights are 21 000, 21 600, 31 000 and 33 000 daltons, respectively. The stoichiometry of the subunits in the native protein is 9 alpha: 9 beta: 2 gamma: 1 delta. R-phycoerythrin carries two covalently linked apoprotein red tetrapyrrol pigments: phycoerythrobilin (PEB) and phycourobilin (PUB). Chemical and spectroscopic data show that alpha subunit carries solely two PEB chromophores, beta subunit--3 PEB and 1 PUB groups, gamma subunit--3 PEB and 2 PUB groups and delta subunit--1 or 2 PEB and 1 PUB groups. The chromophore and polypeptide structure of R-phycoerythrin is mostly composed of all known phycobiliproteins of red and blue-green algae.

[Specific features of intracellular serine proteinase of Bacillus amyloliquefaciens on native and denatured protein substrates]. / Osobennosti deistviia vnutrikletochnoi serinovoi proteinazy Bacillus amyloliquefaciens na nativnye i denaturirovannye belkovye substraty.

The effects of intracellular serine proteinase from B. amyloliquefaciens and of extracellular subtilisin BPN' on native and denaturated protein substrates were compared. The substrate hydrolysis by the enzymes was determined by a method initiating possible participation of intracellular serine protinease in intracellular protein degradation. This approach consists in a prolonged treatment of the products obtained after proteolysis with carboxypeptidase A with a subsequent amino acid assay. It was found that intracellular serine proteinase is capable of degrading denaturated protein substrates e. g. reduced, carboxymethylated ribonuclease A and bovine serum albumin, with an efficiency comparable to that achieved by subtilisin hydrolysis. On the other hand, the intracellular enzyme attacks the native proteins much slower than substilisin.

[Intercellular serine proteinases from spore-forming bacilli]. / Vnutrikletochnye serinovye proteinazy sporoobrazuiushchikh batsill.

Some physico-chemical properties, immunological reactions, structural, functional and evolutionary features of intracellular serine proteinases from spore-forming bacilli were studied. These enzymes belong to an individual subfamily of serine proteinases and in terms of their structure and evolution are most closely related to secretory subtilisins. Intracellular serine proteinases are found in a wide variety of microorganisms--from the spore-forming bacilli to Escherichia coli and are characterized by a higher (as compared to secretory subtilisins) rate of evolution due to a more rigid selective control of the structure and function of intracellular enzymes. Active intracellular proteinases have a unique dimeric structure, which allows to exclude random proteolysis of native proteins in vivo. Secretory subtilisins and intracellular bacillary serine proteinases are coded by separate closely related structural genes, whose presence in the bacillary genome can be explained by duplication of the precursor gene. The existence of duplicated genes of serine proteinases provides sufficient evidence for the marked structural divergence and a high rate of evolution of secretory subtilisins.

[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.

[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.

[Effect of Bacillus subtilis A-50 "streptomycin" mutations on the formation of molecular forms of subtilisin, an extracellular alkaline proteinase]. / Vliianie "streptomitsinovykh" mutatsii Bacillus subtilis A-50 na obrazovanie molekuliarnykh form subtilizina--vnekletochnoi shchelochnoi proteinazy.

The patterns of subtilisin molecular forms of streptomycin-resistant (Strr) and streptomycin-dependent (Strd) mutants of Bacillus subtilis A-50, as well as the revertants of Strd to streptomycin-independence (Str1) were studied. Strr mutants had different quantitative pattern of the same subtilisin molecular forms as compared with the initial strain A-50 (the forms with Rf 0.08, 0.16 and 0.3). In comparison with the initial strain A-50, Strd mutants and Str1 revertants revealed three additional forms of the active enzyme with Rf 0.02, 0.5 and 0.7 and the molecular weights less than 35,000, 28,000 and 20,000 respectively. It was suggested that the rate and character of the enzyme secretion of the degree of its post-translational modifications might result in the different pattern of subtilisin molecular forms produced by these streptomycin mutants.

[Electron microscopic study of Bacillus subtilis mutants differing in the serine proteinase activity and spectrum]. / Elektronno-mikroskopicheskoe issledovanie mutantov Bacillus subtilis, otlichaiushchikhsia po aktivnosti i spektru serinovykh proteinaz.

Vegetative cells and spores of the colonial morphological mutants of Bacillus subtilis A-50 were studied by electron microscopy. The ultrastructure of vegetative cells from both asporogenic colonial-morphological mutants and those which were capable of forming spores in the presence of high concentrations of nitrogen and carbon sources with a decreased activity and a modified spectrum of serine proteases differed from the parent strain by the presence of a microcapsule, the uneven thickness of a cell wall, and the absence of a distinct periplasmic space. Crystalline inclusions of a regular shape were detected in the sporeforming mutant in those cells which were devoid of spores. Spores of the mutant had additional layers.

[Construction and molecular cloning of hybrid plasmids containing specific fragments of Escherichia coli DNA]. / Konstruirovanie i molekuliarnoe klonirovanie gibridnykh plazmid, soderzhashchikh opredelennye fragmenty DNK Escherichia coli.

In the paper a convenient procedure for the isolation of specific Eco RI-fragments of E. coli genome and their amplification on Km-resistance plasmid vector CKdelta11 is described. Plasmid CKdelta11 contains Col E1 replicon and has only one Eco RI site. The hybrid molecules were constructed in vitro using Eco RI-digestion followed by ligation. Then appropriated E. coli strain (polyauxotrophic strain E. coli K12 AB 2463) was transformed with ligated DNA mixture and hybrid plasmids, containing arg, leu, his and thr chromosomal markers were selected by molecular cloning and isolated from the obtained E. coli clones. The hybrid plasmids have two Eco RI sites and consist of one Eco RI-fragment of initial plasmid CKdelta11 and one Eco RI-fragment of El coli DNA. The method described allows to isolate and amplify on hybrid plasmids DNA fragments, containing any selectable genes or genes adjacent to the selectable ones.

[Distribution of DNA sequences recognized by specific endonucleases]. / Raspredelenie posledovatel'nostei DNK, uznavaemykh spetsificheskimi éndonukleazami.

The molecular weight distributions of fragments obtained after endonuclease treatment of DNA were studied. DNA's from pigeon and E. coli and restriction enzymes EcoRI and BamHI were used. The samples of 14C- and 3H-labelled DNA were treated by endonucleases, separated electrophoretically in 1% agatose gels, and radioactivity distributions along gels were measured. From these data weight and number distributions and the average molecular weights of DNA fragments were determined. EcoRI-fragments of phage DNA were used as standards for the molecular weight calibration. The experimental results are compared with the expected data calculated from the DNA GC content. The molecular weight distribution of fragments and the average molecular weights of BamHI-fragments of pigeon DNA and EcoRI- and BamHI-fragments of E. coli DNA differed from random ones. It is suggested that certain genomes contain regions in which the probability of endonuclease cleavage strongly differs from the average probability of such a cleavage for the entire genome.

[Effects of mutations in Bacillus subtilis genome decreasing the protease activity on the formation of subtilisin molecular forms]. / Vliianie mutatsii Bacillus subtilis, snizhaiushchikh proteoliticheskuiu aktivnost; na obrazovanie mnozhestvennykh molekuliarnykh form subtilizina.

Multiple molecular forms of subtilisin--extracellular serine protease produced by the wild strain Bac. subtilis A-50 and its mutant strains with the protease activity decreased two-fold and more were studied. Six molecular forms of subtilisin were found on the whole when 33 mutant strains have been investigated under the experimental conditions. It is essential that both the wild and each of mutant strains under study produced not more than three out of these six forms. Three molecular forms of subtilisin from the mutant strains are similar to those found in the wild strain A-50, and have the molecular weight, of 27 000-30 000. Three other forms of subtilisin were revealed only in the mutant strains, and had the molecular weight of about 20 000. Apparently there is only one structural gene for subtilisin in Bac. subtilis genome. The appearence of multiple molecular forms of subtilisin may be due to the post-translational modifications (limited proteolysis) of the initial type of enzyme, i.e. pre-subtilisin. Probably, that certain mulations not affecting the structural gene can significantly change the expression of such gene by varying of the degree of product modifications.

[Comparative study of the multiplicity of exoenzymes produced by different strains of Bacillus subtilis]. / Sravnitel'noe izuchenie mnozhestvennosti ékzofermentov, produtsiruemykh razlichnymi mutantami Bacillus subtilis.

Molecular forms of two exoenzymes, subtilisin and alpha-amylase, produced by mutants of Bacillus subtilis were studied. The degree of the post-translational modification of subtilisin was less pronounced for P and M mutants than for R mutants. Some of the P and M mutants produced subtilisin having higher molecular weight and hydrophobicity as compared to the R mutants. This form of subtilisin may be the primary product of translation of the structural gene and therefore a precursor of subtilisin. Its appearance outside the cell may be due to the alteration in the cell surface structures in the P and M mutants and abnormal post-translational modification. The P and M mutants produced also differing exocellular proteins as compared to the R mutants, e.g. alpha-amylase forms with the higher isoelectric points.

[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.