[Thermoreactive hydrogels in biotechnology and medicine]. / Termoreaktivnye gidrogeli v biotekhnologii i meditsine.

The properties of thermoreactive hydrogels (TH), i.e., hydrogels formed by polymers which exhibit a lower critical solution temperature (LCST) behaviour in aqueous solutions, are discussed. TH shrink drastically and lose water on heating at temperatures close to LCST resulting in gel collapse. This process is reversible, and TH swell again to their initial size. TH can be successfully used for concentrating suspensions and protein solutions, for immobilization of biocatalysts, in drug delivery systems and in biosensors.

[Effect of physiologically active polymers on the activity and hormonal induction of tyrosine aminotransferase]. / Vliianie fiziologicheski aktivnykh polimerov na aktivnost' tirozinaminotransferazy na ee gormonal'nuiu induktsiiu.

Effect of neutral polymers on activity of tyrosine aminotransferase and hydrocortisone induction of the enzyme were studied. Physiologically active polymers decreased in most cases the basal rate of the enzymatic activity. Preadministration of polymers decreased hormonal induction of tyrosine aminotransferase. Simultaneous administration of polymers and hydrocortisone led to augmented and long-term induction, which appears to occur due to complex-formation of the polymer and hormone. Gradual liberation of hydrocortisone from the complex enabled to maintain the hormone effective concentration for a long time.

[The effect of neutral polymers on glutamate dehydrogenase activity of mouse liver mitochondria upon administration of endotoxin]. / Vliianie neitral'nykh polimerov na aktivnost' glutamatdegidrogenazy mitokhondrii pecheni myshei pri vvedenii éndotoksina.

It is shown that neutral polymers administered intraperitoneally to intact animals considerably affect glutamate dehydrogenase activity in the liver cell mitochondria as well as in the supernatant. Of the tested polymers, only polyvinyl methylacetamide and dextran inhibit a decrease in the level of mitochondrial enzyme activity which develops with administration of endotoxin. Polyvinyl pyrrolidone with molecular weight of 100 kDa, polyvinyl methylacetamide, dextran and polyvinyl caprolactam prevent an increase of glutamate dehydrogenase activity in the supernatant in case of endotoxin administration to animals. It is possibly a result of the effect of the mitochondrial structure stabilization by the above polymers. A physiological effect of polyvinyl pyrrolidone revealed as an effect on the activity level of mitochondrial glutamate dehydrogenase and in the supernatant after endotoxin administration to animals, depends on the molecular weight of the polymer.

[Isolation and properties of aminocaprolactam hydrolase from Providencia alcalifaciens]. / Poluchenie i svoistva aminokaprolaktamgidrolazy iz Providencia alcalifaciens.

L-alpha-aminocaprolactam hydrolase possessing the L-lysinamidase activity was isolated and purified from Providencia alcalifaciens. The purification procedure of enzymes included cell destruction on USDL-1, fractionation by ammonium sulfate, gel-chromatography on G-100, ion exchange chromatography on DEAE-cellulose. The purification resulted in a homogeneous enzyme which possessed the both activities. The enzyme molecular weight (180 kDa) was estimated by gel chromatography on Sephadex G-200. Km was 3.5 mM in the phosphate buffer (pH 7.2). L-alpha-aminocaprolactam hydrolase and L-lysinamidase may be related to metal-dependent enzymes requiring Mg++.

[Aminoacylase from Streptoverticillium microorganisms: stereo- and substrate specificity]. / Aminoatsilaza mikroorganizmov iz roda Streptoverticillium: izuchenie stereo- i substratnoi spetsifichnosti.

The stereo- and substrate specificity of a new aminoacylase from Streptoverticillium microorganisms was studied. The enzyme effectively hydrolyzes acetyl derivatives of aliphatic (methionine, leucine) and aromatic (phenylglycine, phenylalanine, tryptophan) amino acids. The L-enanthiomer of acetylphenylglycine is hydrolyzed by aminoacylase 8000 times more effectively than the D-enanthiomer. A procedure for determination of the enanthioselectivity of aminoacylases was elaborated. This procedure is designed for a detection and assessment of contaminations of the N-acetyl derivative of one enanthiomer by another enanthiomer of the amino acid, as well as of the degree of racemization of the substrate during hydrolysis of acetyl derivatives of D-amino acids.

[Determination of lysine amidase and alpha-aminocaprolactam hydrolase activities in cell-free extracts of Cryptococcus sp]. / Opredelenie lizinamidaznoi i al'fa-aminokaprolaktamgidrolaznoi aktivnostei v beskletochnykh ékstraktakh Cryptococcus sp.

he methods for detecting two activities, i. e. lysine amidase and alpha-aminocaprolactam hydrolase ones, in crude extracts of Cryptococcus sp. are described. The method for registering lysine amidase activity is based on the ability of Cu(II) complex to absorb at 230 nm. The products of lysine and alpha-aminocaprolactam interactions with o-phthalaldehyde in the presence of mercaptoethanol possess different molar absorption at 340 nm. This fact was used for detecting alpha-aminocaprolactam hydrolase activity. The main merit of the methods is the possibility to register the data on the course of the reaction without preliminary chromatographic separation of the reaction products and reactants. The methods proposed do not require expensive enzymes, such as lysine decarboxylase and lysine-alpha-ketoglutarate-epsilon-aminotransferase, which are used for the quantitative estimation of lysine.

[Kinetics and thermodynamics of the hydrolysis-synthesis reaction of acetyl-L-methionine catalyzed by acylase I from hog kidney]. / Kinetika i termodinamika reaktsii gidroliza-sinteza atsetil-L-metionina, kataliziruemoi atsilazoi I suinoi pochki.

The kinetics and thermodynamics of the equilibrium reaction of hydrolysis--synthesis of acetyl-L-methionine catalyzed by acylase I from hog kidney were studied. At high concentrations of the products (acetate ion and L-methionine) the acetyl-L-methionine hydrolysis does not proceed to completion but to the equilibrium position. The equilibrium constant of hydrolysis determined at the attained equilibrium in both directions, i.e. hydrolysis and synthesis, is equal to 3.6 +/- 0.4. Based on the initial rates of hydrolysis and synthesis, a kinetic pattern for the dependence of the reaction rate on concentration of the components of the system is proposed. Evidence for this kinetic pattern is supported by the Holden ratio and the coincidence of the kinetic parameters calculated from the total kinetic curves and the initial rates of hydrolysis and synthesis of acetyl-L-methionine.

[Substrate specificity of acylase I from pig kidney]. / Substratnaia spetsifichnost' atsilazy i svinoi pochki.

The substrate specificity of acylase I from pig kidney has been studied. For the N-acetyl derivatives of D,L-amino acids with an unbranched side chain the catalytic efficiency of enzymatic hydrolysis increases linearly with an increase of the substrate hydrophobicity. The tangent of the linear dependence of logarithm of the second order rate constant of enzymatic hydrolysis on the Hansch hydrophobicity constant is 0,7. This supports a simple "extraction" model of energy realization during the enzyme binding to the substrate in the course of the reaction. When the amino acids with a branched side chain are used, the efficiency of hydrolysis of N-acetyl derivatives decreases by one order of magnitude as compared to the amino acids with an unbranched side chain having the same number of carbon atoms. The N-acetyl derivatives of aromatic amino acids are hydrolyzed in a small degree. An analysis of the substrate specificity of pig kidney acylase I demonstrates that the enzyme has a narrow hydrophobic "cleft" with the length not less than four metrylene links.

[pH dependence of tryptophan ethyl ester hydrolysis]. / PH-zavisimost' nefermentativnogo gidroliza etilovogo efira triptofana.

The dependence of the rate of spontaneous hydrolysis of tryptophan ethyl ester within a wide range of pH (4,6-10,3) was studied. This dependence was found to differ from other dependences, i.e. within the pH range of 4,6-7,0 the value of the rate constant is practically independent on pH. In order to describe the dependence obtained a general pattern of hydrolysis was postulated and the kinetic parameters of individual elementary reactions were determined. The rate constants for the hydrolysis of the amino acid with a non-protonated amino group of ester was calculated using the literary values for the rate constants of carboxylic acid hydrolysis. The obtained values of the second order rate constants for the alkaline hydrolysis of non-protonated and protonated forms of tryptophane ester (k4 and k5) are 1,1 and 79 M-1 s-1, respectively; those for the hydrolysis of the protonated form of the substrate (k3) are 1,0.10(-5) M-1 s-1. The role of spontaneous (non-enzymatic) hydrolysis in stereoselective cleavage of amino acid esters is discussed.

[pH-dependence of tryptophan ethyl ester hydrolysis by alpha-chymotrypsin]. / pH-zavisimost' gidroliza etiolovogo efira triptofana, kataliziruemogo alpha-khimotripsinom.

The hydrolysis of L-tryptophane ethyl ester catalyzed by alpha-chymotrypsin and the effect of ethyl ster of D-tryptophane on the course of the reaction were studied. A kinetic pattern of a three-step enzymatic reaction based on the assumption that the enzyme complex with the protonated form of the substrate is the only reactive one, was proposed. It was shown that the limiting step of the enzymatic reaction consists in a formation of intermediate acyl enzyme. The pH-dependence of the bimolecular rate constant (kcat/Km) for the enzymatic hydrolysis of L-tryptophane ethyl ester is bell-shaped and is described by a pattern including ionization of two groups with pKa values of 7,0 +/- 0,1; and 7,5 +/- 0,1; the value of pKa equal to 7,5 +/- 0,1 corresponds to substrate ionization (pKas for the amino group of L-tryptophane ethyl ester is 7,6). The constants for the binding of protonated and non-protonated substrate forms by the enzyme were calculated from the step-wise dependence of the Km values of pH. An analysis of the bell-shaped dependence of the catalytic constant of enzymatic hydrolysis included determination of pK values of the ionogenic groups of the enzyme--substrate complex (pK'a = 6,8 +/- 0,1 and pK''a = 7,3 +/- 0,1).

[Method of measuring activity of amino acid acylase]. / Metod opredeleniia aktivnosti atsilaz aminokislot.

The paper describes a method to follow acylase activity. The method is based on spectrophotometry of the amino acid released, using o-phthalic aldehyde and mercaptoethanol. The major advantage of the method are its high sensitivity and speed. With the aid of the method kinetic parameters of hydrolysis of N-acetyl-L-methionine and N-acetyl-D,L-methionine catalyzed by pig kidney acylase were determined. Michaelis constants at pH 7.5 were estimated to be 5 +/- 1 and 10 +/- 2, respectively; this being in consistency with the data in the literature. It was shown that N-acetyl-D-methionine, acetate ion and methionine at the concentrations tested (0.01-0.05 M) did not inhibit acylase from a pig kidney.