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A new approach to the regulation of catalytic properties of medically relevant enzymes has been proposed using the novel recombinant preparation of L-asparaginase from Erwinia carotovora (EwA), a promising antitumor agent. New branched co-polymers of different composition based on chitosan modified with polyethylene glycol (PEG) molecules, designated as PEG-chitosan, have been synthesized. PEG-chitosan copolymers were further conjugated with EwA. In order to optimize the catalytic properties of asparaginase two types of conjugates differing in their architecture have been synthesized: (1) crown-type conjugates were synthesized by reductive amination reaction between the reducing end of the PEG-chitosan copolymer and enzyme amino groups; (2) multipoint-conjugates were synthesized using the reaction of multipoint amide bond formation between PEG-chitosan amino groups and carboxyl groups of the enzyme in the presence of the Woodward's reagent. The structure and composition of these conjugates were determined by IR spectroscopy. The content of the copolymers in the conjugates was controlled by the characteristic absorption band of C-O-C bonds in the PEG structure at the frequency of 1089 cm-1. The study of catalytic characteristics of EwA preparations by conductometry showed that at physiological pH values the enzyme conjugates with PEG-chitosan with optimized structure and the optimal composition demonstrated 5-8-fold higher catalytic efficiency (kcat/Km) than the native enzyme. To certain extent, this can be attributed to favorable shift of pH-optima in result of positively charged amino-groups introduction in the vicinity of the active site. The proposed approach, chito-pegylation, is effective for regulating the catalytic and pharmacokinetic properties of asparaginase, and is promising for the development of prolonged action dosage forms for other enzyme therapeutics.

A specific, fast, and easy method for revelation of active plate producers of L-asparaginase using differential medium on the basis of LB or M9 with 1.5% agar was developed. Each 100 ml of LB or M9 medium additionally contained 6-7 ml ofglycerol, 4 g of L-asparagine, 0.2 g of CaCO3, and diagnostic components: 3 ml of 0.2 M CuSO4 x 5H2O and 2.5 ml of 0.1 M K3Fe(CN)6, pH 7.6-7.8. The results were counted 12-20 or 24-48 h after strain growth at 37 degrees C in corresponding mediums. Red color of colonies and colored zone around them showed the ability of the strain under study to destroy asparaginic complexes. The recommended method allows revealing bacterial strains producing L-asparaginase with specific activity of not less than 0.1-3.0 MU/mg of protein.

The receptor properties of influenza virus (IF) isolates/SSSR/90/77 are studied. The isolates are peculiar for losing glycosylation sites (GS) at the Asn131 receptor-binding region (GS131) after passaging in mice and at the Asn158 region (GS158) after cultivation in the presence of mouse serum. The loss of each carbohydrate residue increases the influenza virus affinity for carbohydrate chains with the terminal group Neu5Ac alpha 2-6Gal and reduces its affinity for Neu5Ac alpha 2-3Gal receptors. The effect is more pronounced in the GS158-depleted virus. Upon substitution of asparagine by aspartic acid, the electrostatic component of virus binding to the receptor is altered because of the increased negative charge on hemagglutinin. The virus receptor phenotype changes depending on the cultivation conditions. The isolate adapted to mice has higher affinity to mouse lung cell receptors, while the virus propagated in chick embryos in the presence of inhibitors has higher affinity to allantoic membrane cells.

The review summarizes and analyzes experimental evidence for the properties of glutamine(asparagine)ase from Pseudomonas aurantiaca-548. The enzyme is a tetramer having a molecular weight of 148 kD and consisting of 4 identical subunits having a molecular weight of 37 kD. For glutaminase activity, the optimum pH is in the range of 6.0-8.0, asparaginase activity increases as pH rises. The enzyme is maximally stable at pH 6.8-8.0. The Michaelis constants are 5.3 +/- 0.7 x 10(-6) M for L-glutamine and 5.7 +/- 0.1 x 10(-6) M for asparagine. The reaction products L-aspartate and L-glutamate are competitive inhibitors anazaserine and 6-diase-5-oxo-1-norleucine are classic inhibitors of glutamine(asparagine)ase. The review also presents data on the conditions for culturing Ps. aurantiaca, on the procedures for isolating glutamine(asparagine)ase from biomass of this microbe, on substrate specificity. The results of searching for regulators of catalytic activity, as well as agents enhancing the resistance of enzymes to heat exposures are considered in the paper. Whether the properties of glutamine(asparagine)ase are in conformity with the criteria for primary choice of promising antitumor agents is discussed.

Under conditions of 45-days visual deprivation content of N-acetyl-L-aspartic acid was considerably increased in visual cortex (field 17) tissues and decreased in the superior colliculus and exterior geniculate body. At the same time, content of N-acetyl-L-aspartic acid was not altered in the visual cortex mitochondrial fraction; in the outer structures studied level of this amino acid was distinctly decreased both in tissues and mitochondrial fractions. After prolongation of the visual deprivation up to 90 days content of N-acetyl-L-aspartic acid was sharply decreased in tissues and mitochondrial fractions of all the formations studied of dog brain visual analyzer. The maximal 4.5-fold decrease was found in mitochondrial fractions of the visual analyzer (field 17), in the exterior geniculate body and superior colliculus content of the amino acid was decreased 2.4-3.9-fold as compared with controls. In 90 days-long visual deprivation total activity of phosphate-dependent glutaminase was considerably increased (1.18-3.71-fold as compared with controls) in myelinic (A), membraneous (B), light (C), and heavy synaptosomal and synaptic mitochondrial (E) subfractions of the visual cortex (field 17). The rate of the enzyme activation was distinctly higher in these subfractions as compared with that of the motor and parietal cortical subfractions. After 90 days-long visual deprivation the similar alterations were found in A, B, D and E cerebellum subfractions but these alterations were less distinct as compared with the visual, motor and parietal subfractions.

The effect of thiamine and biotin on the processes of cell division, assimilation of glucose, and accumulation of the biomass and nitrogen in the cells was studied with the Candida yeast. The action of the vitamins depended on the source of nitrogen. In some strains, asparagine can substitute for biotin. Biotin has different effect on the production of gamma-aminobutyric acid in Candida pulcherrima, C. guilliermondii. C. tropicalis K3-10. High concentrations of arginine were found in C. guilliermondii var. membranaefaciens in the presence of biotin. The vitamins did not favour the assimilation of nitrate nitrogen in species which were not adapted to this source of nitrogen.

Asparagine is able to penetrate into human erythrocytes from the external medium. The dependence of the asparagine transport rate on its concentration can be described by the Michaelis-Menten equation with parameters: Km = 2.50 mM, V = 0.24 mmol/l cells per hour. Loading of erythrocytes with asparaginase does not influence their permeability to asparagine. Aspartate is accumulated inside these erythrocytes during incubation with asparagine, thus reflecting rapid transformation of penetrating asparagine by entrapped asparaginase.

The effect of forced cooling was studied as applied to the contents of ammonia, glutamine, glutaminic, asparaginic and alpha-amino butyric acids in the brain of sousliks woken-from hibernation. The cooling of the woken sousliks to the body temperature of 30, 20 and 25 degrees C decreased to some extent the ammonia content in the brain. A deeper hypothermia (10 degrees C) causes its 60,4% decrease as compared to the ammonia amount in woken animals. The cooling of the animals to 30, 25, 20 and 10 degrees C considerably decreases the contents of glutamine, glutaminic acid and GABA in the brain tissue.

It is found that the level of glutamic, aspartic acids as well as of their amides and GABA in tissues located far from the tumour decreases as compared to the control, independently of the tumour strain or the type of the cancerogenic agent. Simultaneously the activities of their metabolic enzymes decrease.

The effect of 12 sources of nitrogen on growth and cholesterine-decomposing activity was studied with Mycobacterium rubrum and Achromobacter candicans. The yield of biomass and the rate of cholesterine decomposition depended on the source of nitrogen and its concentration in the medium. The highest specific activity of the enzyme decomposing cholesterine was found during growth of the cultures on media containing reduced forms of nitrogen. The activity of the enzyme of Mycobacterium rubrum was by 25% higher than that of Achromobacter candicans. The optimum conditions for the production of the enzyme by Mycobacterium rubrum were on media containing 1.0 g of asparagine or 5.0 g of ammonium nitrate per one litre, and for the enzyme production by Achromobacter candicans, on media containing 5.0 g of ammonium phosphate per one litre.

The effect of different nitrogen sources on the biosynthesis of vitamin B12 ano porphyrins by suspensions of resting cells of Propionibacterium shermanii was investigated. In the absence of ammonium nitrogen vitamin B12 was not accumulated by the bacterial cell. This was also the case when its nitrogen containing precursor--delta-aminolevulinic acid was added to the incubation medium. The porphyrins biosynthesis was not changed. When amino acids and their amides were used as the sole nitrogen source, an intensive formation of vitamin B12 developed, if the medium contained aspartic acid, asparagine and cystein subjected to distinct deamination, and glutamine. The study of the composition of extracellular tetrapyrrole compounds showed an accumulation of corrinoil with free carboxyl groups in the incubation medium in the absence of ammonium salts. This corrinoid was identical to cobyrinic acid with respect to its spectral characteristics and behaviour during electrophoresis in phosphate buffer. Possible pathways of the involvement of ammonium nitrogen and glutamine in the biosynthesis of vitamin B12 and the role of amide groups in the binding of corrinoids with cell proteins are discussed.

The content of N-acetyl-l-asparaginic acid, CoA, CoASAc and the N-acetylaspartate amunohydrolase activity was determined in cerebral areas of rats at the normal level and at different stages of oxygen poisoning. At the preconvulsive stage of the oxygen poisoning the content of N-acetyl-l-asparaginic acid decreases in cerebral hemispheres by 54, in the midbrain and diencephalon by 23, in the medulla oblongata--by 27, and in the cerebellum by 21%. The N-acetylaspartate aminohydrolase activity, vice versa, increases by 58, 62, 57 and 60%, respectively. The content of CoA and CoASAc is unchanged. At the convulsive stage of hyperoxia the content of N-acetyl-l-asparaginic acid in the cerebral hemispheres and cerebellum is unchanged in the midbrain and diencephalon increases by 21% and in the medulla oblongata-by 15%. The n-acetylaspartate aminohydrolase activity in he cerebral hemispheres, midbrain, diencephalon and medulla oblongata is unchanged, in the cerebellum it increases by 100%. The CoA content in the brain decreases by 20%, CoASAc-by 16%.

The cells of Pseudomonas fluorescens AG contain two inducable asparaginase enzymes: one of them hydrolyzes only L-asparagine (asparaginase A), the other--L-asparagine, L-glutamine, and D-asparagine (asparaginase AG). In the conditions of continuous cultivation of the bacteria, aspartic and glutamic acids induce the formation of these enzymes only when the amino acids were used simultaneously as a growth-limiting factor and as a sole source of carbon and nitrogen. Both enzymes are not induced in the conditions when the growth is limited by the nitrogen of these amino acids. When the growth was limited by carbon, asparagine, aspartic and glutamic acids induce asparaginase AG more than asparaginase A. Asparagine and glutamine are better inductors than the corresponding amino acids. The activity of asparaginase and glutaminase increases with the specific growth rate of the culture. The induced synthesis of both amidases, after prolonged growth of the culture on a defined medium with glycerol, is inhibited by glycerol but not by glucose. The results are discussed from the viewpoint of regulation of amidases in these bacterial cells.

It is shown that the polypeptide synthetase activity (PS-activity) of chromatin from rat liver is increased 9--21 hrs after partial hepatectomy. Among 9 amino acids studied alanine, methionine, lysine, tyrosine and arginine are not incorporated into the system in question. The highest rate of polymerization is observed in case of glutamic acid. The rate of glutamine, asparagine and glycine incorporation is 7--8 times slower. The PS-activity of chromatin is enhanced by chromatin preincubation with NAD (but not with its analogs). The activation is prevented by thymidine and nicotinamide. Storage of chromatin for 18 hrs at 2--4 degrees C results in a complete loss of PS-activity. Ability of "old" chromatin to incorporate of amino acids may be restored by its preincubation with NAD. Storage of chromatin in the presence of 5 mM cAMP does not decrease the PS-activity. It is assumed that in the system described poly-ADP ribose is an energy source for amino acid activation.

The role of protein amide groups was studied in the process of the organism ageing. It is shown that with ageing there occurs the asparagin deamidation in water-soluble and water-insolbule proteins of the rat brain, liver and heart. The glutamine content in the old rat tissues remains at the level determined in the young animal tissues. Tissue proteins of old rats are better substrates for protein--a complex of proteinases of a wide specificity that those of young animals. An increased attack of the old animals proteins by nonspecific proteinases is due to a decrease in their amidation degree during ageing.

In 58 Citrobacter strains the pathways of the utilization of dicarbonic amino acids and their amides were studied. These organisms were found to be incapable of decarboxylating glutaminic and asparaginic acids, as well as their amides. All the strains could actively desamidizate asparagine. Not all of these strains showed glutaminase activity. Aspartate-aminotransferase occurred twice as often as alanine-aminotransferase, the level of activity being approximately the same. The Citrobacter strains desamidizated asparaginic acid with great constancy, but only in 1/3 of them this reaction occurred via an aspartase route. The desamidization of asparaginic acid in Citrobacter seemed to proceed in different ways. The desamidization of glutaminic acid was observed only in a part of the strains, and the reaction proceeded less actively.

We have studied amino acid up-take from incubation media by colchicine-resistant and colchicine-sensitive Djungarian hamster fibroblast cells. It has been shown that arginine and asparagine amino acid contents in the medium are different for colchicine-sensitive and colchicine-resistant cells. Amino acids concentration is not reduced in the medium after incubation of resistant cells, while it is decreased after incubation of sensitive cells. We can suggest that penetration of low-weight sources of nitrogen into sensitive cells is hampered. It can also be suggested that protein macromolecules are the main source of nitrogen for these cells. The protein up-take levels from the incubation medium, as assessed by the ammonia and amino acid contents of cell counts don't exceed 5% of their initial concentrations in the medium.

The aminoacyl-tRNA-synthetase activity of muscular soluble fraction was studied in normal rabbits, in those fasting for a long period and old rabbits. A rigion of linear dependence is found for the initial rate of the tRNA aminoacylation reaction on the concentration of the enzymic preparation and also on the substrate amount. Having valuated the initial rate of the aminoacylation reaction, the asparagil- and valyl-tRNA-synthetase activities of muscles were determined in normal rabbits, in those fasting for a long period and in old rabbits. The data obtained evidence for an increase of the studied activities in the muscles of the fasting animals and for their decrease in old animals.

The effect of various nitrogen sources (L-glutamic acid, L-glutamine, L-aspartic acid, L-asparagine, and ammonium sulphate) on the synthetase and transferase activity of glutamine synthetase was studied in Candida tropicalis. These nitrogen sources had different effect on the two activity of the enzyme. Glutamic acid or ammonium sulphate did not produce any considerable action on the kinetic properties of glutamine synthetase of this fodder yeast.