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Rodina EV Vorobyeva NN Kurilova SA Belenikin MS Fedorova NV Nazarova TI 《Biochemistry. Biokhimii?a》2007,72(1):93-99
The interaction of Escherichia coli inorganic pyrophosphatase (E-PPase) with effector ATP has been studied. The E-PPase has been chemically modified with the
dialdehyde derivative of ATP. It has been established that in the experiment only one molecule of effector ATP is bound to
each subunit of the hexameric enzyme. Tryptic digestion of the adenylated protein followed by isolation of a modified peptide
by HPLC and its mass-spectrometric identification has showed that it is an amino group of Lys146 that undergoes modification.
Molecular docking of ATP to E-PPase indicates that the binding site for effector ATP is located in a cluster of positively
charged amino acid residues proposed earlier on the basis of site-directed mutagenesis to participate in binding of effector
pyrophosphate. Molecular docking also reveals several other amino acid residues probably involved in the interaction with
effectors.
Published in Russian in Biokhimiya, 2007, Vol. 72, No. 1, pp. 110–117. 相似文献
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Rodina EV Vorobyeva NN Kurilova SA Sitnik TS Nazarova TI 《Biochemistry. Biokhimii?a》2007,72(1):100-108
It has been shown that PPi, methylenediphosphonate, and ATP act as effectors of Escherichia coli inorganic pyrophosphatase (E-PPase), and that they compete for binding at the allosteric regulatory site. On the basis of
chemical modification and computer modeling of a structure of the enzyme-ATP complex, a number of amino acid residues presumably
involved in binding effectors has been revealed. Mutant variants Lys112Gln, Lys112Gln/Lys148Gln, and Lys112Gln/Lys115Ala of
E-PPase have been obtained, as well as a modified variant of wild type E-PPase (Adwt PPase) with a derivative of ATP chemically attached to the amino group of Lys146. Kinetic properties of these variants
have been investigated and compared to the earlier described variants Lys115Ala, Arg43Gln, and Lys148Gln. Analysis of the
data confirms the proposed location of an effector binding site in a cluster of positively charged amino acid residues including
the side chains of Arg43, Lys146 (subunit A), Lys112, and Lys115 (subunit B). Lys112 is supposed to play a key role in forming
contacts with the phosphate groups of the three studied effectors.
Published in Russian in Biokhimiya, 2007, Vol. 72, No. 1, pp. 118–127. 相似文献
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L. S. Kurilova Z. I. Krutetskaya O. E. Lebedev V. G. Antonov 《Cell and Tissue Biology》2008,2(3):322-332
Using Fura-2AM microfluorimetry, the effect of oxidized glutathione (GSSG) and its pharmacological analogue glutoxim on the intracellular Ca2+ concentration in rat peritoneal macrophages was investigated. It was shown that GSSG or glutoxim increase the intracellular Ca2+ concentration by inducing Ca2+ mobilization from thapsigargin-sensitive Ca2+ stores and subsequent Ca2+ entry from external medium. Dithiothreitol, which reduces S-S-bonds in proteins, completely prevents or reverses the increase of intracellular Ca2+ concentration induced by GSSG or glutoxim. This suggests that the increase of intracellular Ca2+ concentration induced by GSSG or glutoxim can be mediated by their interactions with functionally important SH-groups of proteins involved in Ca2+-signaling.Two structurally different tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate prevent or completely reverse the increase in the intracellular Ca2+ concentration induced by GSSG or glutoxim. On the contrary, tyrosine phosphatase inhibitor Na orthovanadate enhances the increase of intracellular Ca2+ concentration evoked by oxidizing agents. The data suggest that tyrosine kinases and tyrosine phosphatases are involved in the regulatory effect of GSSG and glutoxim on the intracellular Ca2+ concentration in macrophages. 相似文献
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Moiseev VM Rodina EV Kurilova SA Vorobyeva NN Nazarova TI Avaeva SM 《Biochemistry. Biokhimii?a》2005,70(8):858-866
Escherichia coli inorganic pyrophosphatase (PPase) is a one-domain globular enzyme characterized by its ability to easily undergo minor structure rearrangements involving flexible segments of the polypeptide chain. To elucidate a possible role of these segments in catalysis, catalytic properties of mutant variants of E. coli PPase Gly100Ala and Gly147Val with substitutions in the conservative loops II and III have been studied. The main result of the mutations was a sharp decrease in the rates of conformational changes required for binding of activating Mg2+ ions, whereas affinity of the enzyme for Mg2+ was not affected. The pH-independent parameters of MgPP(i) hydrolysis, kcat and kcat/Km, have been determined for the mutant PPases. The values of kcat for Gly100Ala and Gly147Val variants were 4 and 25%, respectively, of the value for the native enzyme. Parameter kcat/Km for both mutants was two orders of magnitude lower. Mutation Gly147Val increased pH-independent Km value about tenfold. The study of synthesis of pyrophosphate in the active sites of the mutant PPases has shown that the maximal level of synthesized pyrophosphate was in the case of Gly100Ala twofold, and in the case of Gly147Val fivefold, higher than for the native enzyme. The results reported in this paper demonstrate that the flexibility of the loops where the residues Gly100 and Gly147 are located is necessary at the stages of substrate binding and product release. In the case of Gly100Ala PPase, significant impairment of affinity of enzyme effector site for PP(i) was also found. 相似文献
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Kurilova AA Proskurina VA Maĭskaia VD 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》2004,(3):81-83
The homogeneity of colonies of two B. anthracis vaccine strains in R- and RS- forms (100 colonies of each strain) in terms of their adhesive capacity was studied. B. anthracis strain 228/8 showed more heterogeneous composition than B. anthracis strain 71/12, moderately and highly adhesive colonies prevailing in both forms and nonadhesive colonies being absent. The prevalence of highly adhesive clones was established in the RS- form of B. anthracis strain 72/12 in comparison with R- form. By the average value of the adhesion index the RS- form colonies of this strain were classified as highly adhesive, while the colonies in the R- form were characterized as moderately adhesive. 相似文献
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Glutoxim and molixan belong to new generation of disulfide-containing drugs with immunomodulatory, hepatoprotective and hemopoetic effect on cells. Using Fura-2AM microfluorimetry, two structurally distinct actin filament disrupters, latrunculin B and cytochalasin D, and calyculin A, which causes actin filaments condensation under plasmalemma, we have shown the involvement of actin cytoskeleton in the intracellular Ca(2+)-concentration increase induced by glutoxim or molixan in rat peritoneal macrophages. Morphological data obtained with the use of rhodamine-phalloidine have demonstrated that glutoxim and molixan cause the actin cytoskeleton reorganization in rat peritoneal macrophages. 相似文献
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Rodina EV Vainonen LP Vorobyeva NN Kurilova SA Sitnik TS Nazarova TI 《Biochemistry. Biokhimii?a》2008,73(8):897-905
Inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) is one of the possible targets for the rational design of anti-tuberculosis agents. In this paper, functional properties of this enzyme are characterized in the presence of the most effective activators--Mg2+ and Mn2+. Dissociation constants of Mt-PPase complexed with Mg2+ or Mn2+ are essentially similar to those of Escherichia coli PPase. Stability of a hexameric form of Mt-PPase has been characterized as a function of pH both for the metal-free enzyme and for Mg2+- or Mn2+-enzyme. Hexameric metal-free Mt-PPase has been shown to dissociate, forming monomers at pH below 4 or trimers at pH from 8 to 10. Mg2+ or Mn2+ shift the hexamer-trimer equilibrium found for the apo-Mt-PPase at pH 8-10 toward the hexameric form by stabilizing intertrimeric contacts. The pK(a) values have been determined for groups that control the observed hexamer-monomer (pK(a) 5.4), hexamer-trimer (pK(a) 7.5), and trimer-monomer (pK(a) 9.8) transitions. Our results demonstrate that due to the non-conservative amino acid residues His21 and His86 in the active site of Mt-PPase, substrate specificity of this enzyme, in contrast to other typical PPases, does not depend on the nature of the metal cofactor. 相似文献