Identification of functional regions in the Rhodospirillum rubrum L-asparaginase by site-directed mutagenesis.

Site-directed mutagenesis of Rhodospirillum rubrum L-asparaginase (RrA) was performed in order to identify sites of the protein molecule important for its therapeutic and physico-chemical properties. Ten multipoint mutant genes were obtained, and five recombinant RrA variants were expressed in E. coli BL21(DE3) cells and isolated as functionally active highly purified proteins. Protein purification was performed using Q-Sepharose and DEAE-Toyopearl chromatography. Overall yield of the active enzymes was 70-80 %, their specific activity at pH 7.4 and 37 °C varied of 140-210 U/mg. L-Glutaminase activity did not exceed 0.01 % of L-asparaginase activity. All RrA mutants showed maximum enzyme activity at pH 9.3-9.5 and 53-58 °C. Km and Vmax values for L-asparagine were evaluated for all mutants. Mutations G86P, D88H, M90K (RrAH), G121L, D123A (RrАI) caused the loss of enzyme activity and confirmed the importance of these sites in the implementation of catalytic functions. Removal of four residues from C-terminal area of the enzyme (RrAK) resulted in the enzyme instability. Mutations D60K, F61L(RrАD), and R118H, G120R(RrАJ) led to the improvement of kinetic parameters and enzyme stabilization. Substitutions E149R, V150P (RrАB) improved antineoplastic and cytotoxic activity of the RrA. A64V, E67K substitutions, especially in combination with E149R, V150P (RrАE), considerably destabilized recombinant enzyme.

[Determination of protease activity in blood and in microorganisms].

For determination of protease activity it is possible to use immunoglobulins. Since proteolytic products apparently do not retain substrate antigenic determinants, it is possible to use ELISA methodsfor monitoring for enzymatic process. ELISA determination of functional activity of specific IgA1-protease has been applied not only for detection of this enzyme, but also for measurement of its inhibition constants. Fixed on a micropanel IgG may be used for evaluation of total proteolytic activity. Depending on pH values, it is possible to measure activity of neutral, alkaline and acid proteases. This approach has allowed to estimate total proteolytic activity of neutral proteases of serum. Measurement of a total level of serum pepsinogene activity can have diagnostic importance in gastroenterology, due to decisive contribution of pepsinogen I to the measured activity.

[A drop of mink complement kills a mouse]. / Kaplia komplementa norok ubivaet mysh'.

The phenomenon of fast death of mice after parenteral administration of mink serum was explained by high activity of mink complement in particular by unusually high activity of its alternative pathway of activation. The presence of antibodies to mouse erythrocytes in mink serum was necessary precondition for their lysis under action of mink complement by classical and alternative pathways. However, removal of these antibodies resulting in cancellation of hemolysis did not effect toxicity of mink serum for nice in vivo. Partial decomplementization of mink serum zymosan completely prevented death of animals.

[Multiple modality treatment of patients with senile central chorioretinal dystrophy with use of electromagnetic field]. / Kompleksnoe lechenie bol'nykh s senil'noi tsentral'noi khorioretinal'noi distrofiei setchatki s primeneniiem élektromagnitnogo polia.

Fifty-one elderly patients with maculodystrophy were observed and treated. Exposure to electric field of 1-2 mV/cm and 40-57 Hz led to improvement of microcirculation in the fundus oculi and decreased vision disorders.

[Activation of kallikrein-kinin system, degranulating activity of neutrophils and blood-brain barrier in schizophrenia]. / Aktivatsiia kallikrein-kininovoi sistemy, degranuliatsionnaia aktivnost' neitrofilov i gematoéntsefalicheskii bar'er pri shizofrenii.

To evaluate permeability of blood-brain barrier (BBB) some immunological and biochemical indices were used. The levels of the activity of serum kallikrein-kinin system (KKS), compliment system, C-reactive protein (CRP) concentration, blood inhibitory potential, metabolic and degranulating activities of neutrophils, as well as functional activity of leukocytic elastase were investigated in 30 patients. Acute schizophrenic attack was accompanied by both activation of KKS and by the increase of functional activity of alfa-1-proteinase inhibitor. The increase of CRP levels, high hemolytic activity of complement as well as considerable degranulating activity of polymorphonuclear leukocytes may be the causes of the damage of BBB permeability during acute schizophrenic attack.

Spatial speckle correlometry in applications to tissue structure monitoring.

Asymptotic behavior of temporal autocorrelation functions of speckle intensity fluctuations induced by tissue scanning with a focused probe beam is experimentally studied for the transition from a single-scattering to a multiple-scattering mode. Such parameters as the exponential factor (or the Hurst coefficient) and the Hausdo dimension are proposed for the characterization and the visualization of the variations of the studied tissues' optical properties in generalized form. We studied reversible transition between various scattering modes stimulated by the application of certain chemical agents to the human sclera samples using speckle intensity correlation analysis; corresponding results are presented. The possibilities of the scattering structures imaging with local estimations of the exponential factor of speckle intensity fluctuations are shown in in vitro experiments with samples of human skin epidermis.

[Study of phage T7 DNA-dependent RNA-polymerase using GTP analogs. Affinity modification and study of interaction with matrices using fluorescent markers]. / Issledovanie DNK-zavisimoi RNK-polimerazy faga T7 s pomoshch'iu analogov GTP. Affinaia modifikatsiia fluorestsentnymi metkami i issledovanie vzaimodeistviia s matritsei.

Interactions of the bacteriophage T7 DNA-dependent RNA polymerase with three GTP analogs have been studied. All of the three analogs tested contained substituted naphthalenesulphamide groups and were shown to be under appropriate conditions irreversible covalent inhibitors of the enzyme, the modified enzyme possessing fluorescent properties. One of these analogs contained the reactive 2-bromoethyl phosphonate group and was shown to cause the loss of the enzyme affinity for polynucleotide templates. The other two modifiers which contained the azide reactive group did not alter the enzyme-template affinity, the polynucleotide binding leading to a notable increase of the enzyme fluorescence intensity. The latter two modifiers are supposed to be convenient for fluorescent labelling of the active site of RNA polymerase for enzyme-template binding studies.

[Affinity modification of DNA-dependent RNA-polymerase of phage T7 with 5'-p-fluorosulfonylbenzoyladenosine]. / Affinnaia modifikatsiia DNK-zavisimoi RNK-polimerazy faga T7 5'-p-ftorsul'fonilbenzoiladenozinnom.

The affinity modification of the DNA-dependent RNA-polymerase of bacteriophage T7 was carried out by using the specific irreversible inhibitor, 5'-p-fluorosulfonylbenzoyladenosine. The inhibitor was found to bind to the enzyme's active site; the kinetic constants of the modification were calculated. The stoichiometry of the covalent E.I-complex formed was determined by using the 14C-labeled inhibitor.

Catalytic activity and association of pancreatic lipase.

The authors summarize their work concerning the mechanism of pancreatic lipase activation. The activation of lipase by submicellar SDS concentrations was found to imitate closely enough its activation by an interface. Lipase activation was shown to be caused by changes in the rate constants for substrate chemical transformation and to involve conformational changes of the enzyme and its association. The complex of a conformationally modified lipase with the detergent, which acts as a 'structure-forming' agent, is associated with native lipase molecules setting up their active site. The mechanism of lipase activation at an interface both in vitro and in vivo is discussed.

[Activation of pancreatic lipase by detergents. III. Oligomerization of the enzyme]. / Aktivatsiia pankreaticheskoi lipazy detergentami. III. Oligomerizatsiia fermenta.

Activation of pancreatic lipase by non-micellar solution of sodium dodecylsulphate (SDS) has been studied. By means of gel-filtration it was found that SDS forces the lipase to form an octamer. A new method of the active sites titration using alkylboronic acids is proposed. The octameric form of the lipase was shown to contain six active sites at the optimal SDS concentration. The activated form of pancreatic lipase supposedly contains six native subunits, each of them forming an active site, and two conformationally altered subunits. This model was confirmed by a probability-theoretic calculation.

[Conformational states of pancreatic lipase]. / Konformatsionnye sostoianiia pankreaticheskoi lipazy.

Spin-label method was applied to the studies of conformation properties of pancreatic lipase. Spin-labelled derivatives of the enzyme in SH- and NH2-groups were obtained. ESR-spectra of both samples belong to the immobilized type, in the first case the ESR-spectrum corresponding to strong immobilization of the spin-label, and in the second--to the average one. In both cases the rotation correlation time of the enzyme molecule was measured. The time proved the same independent of the site of the label attachment; it corresponded to the rotation of macromolecule with molecular weight 50000. This fact points to the absence of both intramolecular flexibility of the enzyme molecule and of the association of lipase molecules in solution. It has been shown that introduction of substrates and inhibitors of the enzyme and the interface as well, induces no changes in the ESR spectra, which points to the absence of local conformation changes of protein near the spin-labels introduced.