[Hydrolysis of chimeric proteins by enteropeptidase at the specific linker (Asp)4Lys depending on refolding conditions]. / Osobennosti gidroliza khimernykh belkov énteropeptidazoi po spetsificheskomu linkeru (Asp)4Lys v zavisimosti ot uslovii refoldinga.

Refolding from inclusion bodies of chimeric proteins containing the enteropeptidase-specific linker (Asp)4Lys was carried out. It was shown that, depending on the refolding conditions, chimeric proteins function as substrates or inhibitors of the enteropeptidase. The efficiency of the enteropeptidase hydrolysis of chimeric proteins containing the (Asp)4Lys linker may depend not only on the amino acid sequence of the protein binding site for the enzyme but also on the site conformation.

[Duodenase--a potential activator of cascade of digestive proteases]. / Duodenaza--potentsial'nyi aktivator kaskada pishchevaritel'nykh proteinaz.

The substrate specificity of duodenase from bovine duodenum mucosa to synthetic and natural polypeptides was studied. Amino acid residues preferential for duodenase in the P1 and P2 positions of the substrate were determined. It was shown that the enzyme is synthesized in epithelial secretory cells of duodenal (Brunner's) glands and enters, as part of the secreta, into the lumen of the duodenum. The possible role of duodenase as an activator of proenteropeptidase is discussed.

[Digestion of Luliberine analogues containing D-alanine residue by human gastric juice]. / Rasshcheplenie zheludochnym sokom cheloveka analogov liuliberina, soderzhashchich ostatok D-alanina.

The digestion of surphagone and other luliberine analogues containing residues of D-amino acids by human gastric juice was studied. By means of chromatography and mass spectrometry, these peptides were shown to undergo hydrolysis of the bond formed by D-Ala at the P-1 position, and this hydrolysis was shown to be catalyzed by gastricsin rather than pepsin A. Gastricsin was assumed to be a highly specific protease. The results are in a good agreement with the concept of conformational specificity of proteases towards the natural oligopeptides.

[Effectiveness of proteolytic enzymes]. / Effektivnost' proteoliticheskikh fermentov.

A theory of the ground state's destabilization is developed explaining the high efficiency of proteases. The theory suggests a participation of the valence interactions in the formation of a productive complex and connects the efficiency and the specificity of proteolysis with the structure of such a complex. Proved is nonproductivity of nonvalence complexes in which the reactive amide group of the substrate is planar. It is suggested that the theory of the absolute rates of reactions does not cover the process of the enzymatic hydrolysis.

[Theoretical aspects of the mechanism of proteolytic enzyme action. V. C-terminal specificity of serine proteinases]. / Teoreticheskie aspekty mekhanizma deistviia proteoliticheskikh fermentov. V. C-kontsevaia spetsifichnost' serinovykh proteinaz.

Tetrahedral intermediates of the alpha-chymotrypsin acylation by N-protected amide dipeptide substrates having different leaving groups were analyzed by means of the molecular mechanic method. A predominant role of the C-terminal interactions in the leaving group protonation and in the development of a kinetic specificity was determined. The kinetic specificity of serine proteases was shown to be controlled by the transformation of various forms of tetrahedral substrate intermediates at the presteady stage.

[Quantum chemistry analysis of the mechanism of action of proteolytic enzymes. III. Proton transport in serine proteases]. / Kvantovo-khimicheskii analiz mekhanizma deistviia proteoliticheskikh fermentov. III. Perenos protona v proteazakh serinovogo tipa.

The model system for the proton transfer on the amide atom of the substrate leaving group based on the existence of "charge relay system" in the serine type proteases was analysed by the CNDO/2 method. The unfitness of this model to explain the action mechanism of serine proteases was shown. The model system for proton transfer with the water molecule as the intermediate acceptor of the Ser-195 proton was suggested and analysed by the same method. The acylation activation barrier of this system was shown to localize on the stage of synchronous transfer of the Ser-195 alcoholic proton and the water molecule proton hydrogen bound to the His-57 N epsilon 2-atom on the water molecule oxygen atom and the N epsilon 2-atom, respectively. The protonation of substrate in the case of the model system with the water molecule as the intermediate acceptor of proton was demonstrated to begin before the completion of the tetrahedral intermediate substance and the protonated from of the tetrahedral intermediate was shown to form only. A hypothesis considering the role of this water molecule as the nucleophilic reagent on the deacylation stage is presented.

[Quantum chemistry analysis of the mechanism of action of proteolytic enzymes. I. The state of the cleavable bond of substrates and intermediates]. / Kvantovo-khimicheskii analiz mekhanizma deistviia proteoliticheskikhfermentov. I. Sostoianie rasshchepliaemoi sviazi substratov i promezhutochnykh soedinenii.

Electronic parameters of amide and ester bonds in some compounds, modelling substrates of proteolytic enzymes, and electronic properties of corresponding tetrahedral compounds, which are intermediates of the hydrolytic reaction, were calculated by the CNDO/2 method. The nature of substituents and the formation of the hydrogen bond by the carbonyl oxygen atom were shown to have no sufficient influence on the charges and bond orders of the amide group. The dramatic dependence of the amide electronic state from the distort degree of its planar structure was found. The resonance stabilization was shown to be absent in the bicyclic beta-lactams. The pK alpha values of the amide nitrogen atom were calculated at various hybridization states in amides.

[Quantum chemistry analysis of the mechanism of action of proteolytic enzymes. II. Nucleophilic attack]. / Kvantovo-khimicheskii analiz mekhanizma deistviia proteoliticheskikhfermentov. II. Nukleofil'naia ataka.

Dynamic systems, modelling the elementary act of nucleophilic attack on the carbonyl carbon atom of amide (N-methyl-acetamide) and ester (methylacetate) substrates by some compounds, simulating the nucleophilic group of various types proteases active site were calculated and analysed by the CNDO/2 method, namely: 1) methoxyanion and the methanol (serine proteases), 2) water molecule in the presence of formate anion (carboxylic proteases) and 3) mercaptide anion (CH3S-) (thiolic proteases). The formation of productive enzyme-substrate complex was shown not only to orient reactive groups of the enzyme and substrate, but also to activate it by induction of a certain degree of cleavable pyramidalization, as a result of the partial resonance stabilization energy loss.

[Determination of leucine-binding protein content of Escherichia coli cells]. / Metody testirovaniia leitsinsviazyvaiushchego belka i opredelenie ego soderzhania v kletkakh E. coli.

A radioimmunochemical method of determination of leucine-binding protein and a method, based on the selective absorption of the protein and its complex with leucine on DEAE-cellulose, has been developed. The protein content in the E. coli cells at different stage of growth has been determined by the radioimmunochemical and equilibrium dialysis methods. It was shown that the protein content in the cells is practically independent of the growth-phase.