[Catalytic properties of Rhodotorula aurantiaca KM-1 phenylalanine ammonia-lyase].

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) of the Rhodotorula aurantiaca strain KM-1 deaminates L-phenylalanine according to the Michaelis-Menten kinetics with K(M) = 1.75 +/- 0.44 mM and V(max) = 3.01 +/- 0.43 units/mg. The enzyme is competitively inhibited by D-phenylalanine with K(in) = 3.38 +/- 0.32 mM. The Michaelis-Menten kinetics was analyzed, the inhibition type (competitive, noncompetitive, and mixed) was identified, and corresponding kinetic parameters were calculated using the computer programs written in Gauss 4.0. PAL was most stable at pH 6.55 and lacked approximately 50% of its activity after incubation at 57 degrees C for 15 min. The yield of L-phenylalanine increased in the presence of mercaptoethanol, sodium ethylenediaminetetraacetate (EDTA), and ascorbic acid. The effects of EDTA and ascorbic acid were additive.

[Transgenic Tobacco plants expressing bacterial genes encoded the thermostable glucanases]. / Transgennye rasteniia tabaka, ékspressiruiushchie bakterial'nye geny termostabil'nykh gliukanaz.

It is shown that bacterial genes for thermostable beta-glucanases are expressed retaining their activity and substrate specificity. The leader peptide of the carrot extensin exerts effective secretion of the bacterial enzymes into the intercellular space of the plant tissue. Expression of the bacterial gene for beta-1,3-glucanase in plant tissues alters their morphogenetic potential. Regeneration of shoots from the calli of these plant lines requires a six- to eightfold increase in cytokinin (6-BAP) concentration in comparison with the control lines and the transgenic lines expressing beta-1,3-1,4-glucanase. Rooting of transgenic plants expressing the bacterial gene for beta-1,3-glucanase occurs much faster. The transgenic plants obtained in the study are proposed as model objects for investigating the role of glucanases in plants.

[Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. I. Construction of vector plasmids for transfer into plants and expression of a modified gene for beta-1,3-glucanase from Clostridium thermocellum in tobacco protoplasts]. / Konstruirovanie i analiz transgennykh rastenii Nicotiana tabacum L. s ékspressiruiushchimsia bakterial'nym genom beta-1,3-gliukanazy. I. Konstruirovanie vektorynykh plazmid dlia perenosa v rasteniia i ékspressiia modifitsirovannogo gena beta-1,3-gliukanazy iz Clostridium thermocellum v protoplastakh tabaka.

We constructed two vectors, pC27-glc and pC29-glc, that allow expression of the beta-1,3-glucanase gene (glc) in plant cells. The glc gene was previously cloned from anaerobic thermophilous bacterium Clostridium thermocellum. To increase the efficiency of expression, the N-terminal fragment of the glc gene encoding bacterial transient peptide was deleted, and hybrid variants of lacZ-glc were obtained. Analysis of expression of the hybrid genes in Escherichia coli showed that deletion of the fragment corresponding to 31 amino acids (a.a.) of beta-glucanase affected neither activity nor thermostability of the enzyme. The modified gene was subcloned into two vectors, pC27 and pC29, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter from Arabidopsis, respectively. Each of the resulting plasmids, pC27-glc and pC29-glc, was transfected into protoplasts of Nicotiana plumbaginifolia. Both the plasmids were shown to allow a high level of activity of the thermostable beta-1,3-glucanase. We plan to use the vectors obtained for transformation of agrobacteria and construction of transgenic plants.

[Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. II. Transgenic tobacco plants expressing the bacterial beta-glucanase gene from Clostridium thermocellum,--a model for studying the differential expression of stress response-related genes]. / Konstruirovanie i analiz transgennykh rastenii Nicotiana tabacum L. s ékspressiruiushchimsia bakterial'nym genom beta-1,3-gliukanazy. II. Transgennye rasteniia tabaka, ékspressiruiushchie bakterial'nye gen beta-gliukanazy iz Clostridium thermocellum,--model' dlia izucheniia differentsial'noi ékspressii genov stressovogo otveta.

The modified hybrid beta-1,3-glucanase gene (glc) of Clostridium thermocellum was expressed in tobacco Nicotiana tabacum. The glc gene was cloned into two plasmids, pC27-glc and pC29-glc, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter of Arabidopsis, respectively. These constructions were used for transformation of agrobacteria followed by transfer into plants. In transformed plants, each plasmid caused a high level of activity of thermostable bacterial glucanase not observed in reference plants. The plants obtained were used to study activation of some defense-related genes induced by their interaction with either tobacco mosaic virus (TMV) or a pathogenic fungus.

[The effect of L-glutamic acid on the ADP-induced aggregation and Ca, Mg-ATpase activity of human thrombocytes]. / Vliianie L-glutaminovoi kisloty na ADF-indutsirovannuiu agregatsiiu i aktivnost' Ca, Mg-ATFazy trombotsitov cheloveka.

In the study of L-glutaminic acid effects on human platelet functional activity it was established that 30-60-min treatment of platelet-rich plasma with glutamate in concentration 0.1-1.0 mM at 37 degrees C results in a marked platelet activation. The latter noticeably enhances ADP-induced aggregation persistent against antiaggregation adenosine concentrations. With increasing duration of the incubation a glutaminic acid minimal effective concentration lowers. Proaggregatory and anti-adenosine effects of L-glutaminic acid are inhibited by kainic acid. The experiments evidence that 30-min preincubation of platelet membranes with 1 mM L-glutaminic acid produces inhibition of cAMP-induced activation of Ca, Mg-ATPase returning the activity of the enzyme to baseline levels. No glutamate-related changes in calcium pump of platelets are reported.

[The regulation of protein kinase C activity in normal, immortalized and transformed rat fibroblasts]. / Reguliatsiia aktivnosti proteinkinazy C v normal'nykh, immortalizovannykh i transformirovannykh fibroblastakh krysy.

In view of recent studies showing that cell proliferation of E1Aad5+c-Ha-ras-transformed fibroblasts cannot be regulated by growth factors and phorbol eaters in contrast to normal and E1Aad5-immortalized cell lines, the present work was undertaken to examine the role of protein kinase C (PKC) in the mitogenic signal transduction machinery in rat embryonal fibroblasts. It is shown that PKC is activated by acidic growth factor and phorbol esters in all the three cell lines. These findings suggest the existence of an additional, not associated with PKC-, growth-signaling pathway in E1Aad5-Ha-ras-transformed rat embryonal fibroblasts.

[Glutamate-binding membrane proteins from human platelets]. / Glutamatsviazyvaiushchie membrannye belki trombotsitov cheloveka.

Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.

[A rapid method of isolation of platelet membranes for radioligand and enzymatic assays]. / Bystryi metod polucheniia kletochnykh membran trombotsitov dlia radioligandnykh i énzimaticheskikh issledovanii.

A simple and rapid procedure for isolation of the total platelet membrane fraction by chromatography on Sepharose CL 6B has been developed. This method allows a rapid (25-30 min) one-step separation of the membrane fraction on 26 x 150 mm columns with a 20-21 mg recovery. The high degree of purity of membrane preparations was confirmed by a radioligand assay using [3H]adenosine and L-[3H]glutamic acid. The purity of membrane preparations is comparable with that of membrane fractions obtained by standard ultracentrifugation methods. The homogeneity of the membrane fraction was demonstrated by using marker enzymes of plasma, microsomal and mitochondrial membranes. This finding is very important in that it allows the isolation of fractions differing in their protein content with no effect on the method reproducibility. The high utility of the membrane preparations in receptor studies was demonstrated for high affinity binding sites for [3H]adenosine and L-[3H]glutamic acid.

[The sites of high affinity binding of L-[3H]glutamic acid in human platelets. A new type of platelet receptor?]. / Uchastki vysokoaffinnogo sviazyvaniia L-[3H]glutaminovoi kisloty v trombotsitakh cheloveka. Novyi tip trombotsitarnogo retseptora?

The total membrane fraction of human platelets was found to contain high affinity sites of L-[3H]glutamic acid binding (Kd = 100 nM, Bmax = 1.06 pmol/mg protein). The pH optimum for binding is at pH approximately 6.9 Na+ (1-150 mM) inhibit glutamate binding by platelet membranes (IC50 = 12 mM). Ca2+ (50-100 microM) stimulate the binding by 10-20% and inhibit it by 20-30% at concentrations of 1-5 mM. Monoclonal antibodies to the glutamate receptor strongly suppress the L-[3H]glutamate binding by platelet membranes (IC50 = 300 nm). The presence in human platelets of a glutamate-sensitive receptor complex similar to the central nervous system glutamate receptor is postulated.