A new approach to improving the accuracy of elemental analysis in laser mass spectrometry.

It is shown that the main reason affecting the accuracy and reproducibility of the elemental analysis of solids with a laser source of ions is the difference in the ionization cross sections of different elements in the generation of laser plasma. Computer modeling was carried out for the evaporation of the sample and generation of laser plasma at different values of laser power density. The aim of modelling was to determine the cause of the low accuracy of analysis by direct selection of ions from the plasma. The researches have shown that we cannot get satisfactory accuracy of analysis using analytical signal formed only on the base of single-charged ions. At the same time, in the formation of an analytical signal, as a sum of the intensities of the mass peaks of all charges of each element, the accuracy of the analysis does not depend either on the ionization cross section or on the nonreproducibility of the laser radiation power. It is shown that this approach completely eliminates the "matrix effect."

Expression, purification and crystallization of a thermostable short-chain alcohol dehydrogenase from the archaeon Thermococcus sibiricus.

Alcohol dehydrogenases belong to the oxidoreductase family and play an important role in a broad range of physiological processes. They catalyze the cofactor-dependent reversible oxidation of alcohols to the corresponding aldehydes or ketones. The NADP-dependent short-chain alcohol dehydrogenase TsAdh319 from the thermophilic archaeon Thermococcus sibiricus was overexpressed, purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion method using 25%(w/v) polyethylene glycol 3350 pH 7.5 as precipitant. The crystals diffracted to 1.68 A resolution and belonged to space group I222, with unit-cell parameters a = 55.63, b = 83.25, c = 120.75 A.

Crystallization and preliminary X-ray diffraction analysis of Salmonella typhimurium uridine phosphorylase complexed with 5-fluorouracil.

Uridine phosphorylase (UPh; EC 2.4.2.3) catalyzes the phosphorolytic cleavage of the N-glycosidic bond of uridine to form ribose 1-phosphate and uracil. This enzyme also activates pyrimidine-containing drugs, including 5-fluorouracil (5-FU). In order to better understand the mechanism of the enzyme-drug interaction, the complex of Salmonella typhimurium UPh with 5-FU was cocrystallized using the hanging-drop vapour-diffusion method at 294 K. X-ray diffraction data were collected to 2.2 A resolution. Analysis of these data revealed that the crystal belonged to space group C2, with unit-cell parameters a = 158.26, b = 93.04, c = 149.87 A, alpha = gamma = 90, beta = 90.65 degrees . The solvent content was 45.85% assuming the presence of six hexameric molecules of the complex in the unit cell.

[A comparative structure-function analysis and molecular mechanism of action of endonucleases from Serratia marcescens and Physarum polycephalum]. / Sravnitel'nyi strukturno-funktsional'nyi analiz éndonukleaz Serratia marcescens i Physarum polycephalum i molekuliarnyi mekhanizm ikh deistviia.

Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water-magnesium complex involved in their catalytic acts. In the enzyme-substrate complexes, the Mg2+ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3'-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3'-leaving group. A strained metalloenzyme-substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction. The English version of the paper.

[X-ray structural analysis of magnesium-containing Serratia marcescens endonuclease]. / Rentgenostrukturnyi analiz magniisoderzhashchei éndonukleazy Serratia marcescens.

The three-dimensional crystal structure of the DNA/RNA nonspecific endonuclease from Serratia marcescens was refined at the resolution of 1.07 A to R factor of 12.4% and Rfree factor of 15.3% using the anisotropic approximation. The structure includes 3924 non-hydrogen atoms, 715 protein-bound water molecules, and a Mg2+ ion in each binding site of each subunit of the nuclease homodimeric globular molecule. The 3D topological model of the enzyme was revealed, the inner symmetry of the monomers in its N- and C-termini was found, and the local environment of the magnesium cofactor in the nuclease active site was defined. Mg2+ ion was found to be bound to the Asn119 residue and surrounded by five associated water molecules that form an octahedral configuration. The coordination distances for the water molecules and the O delta 1 atom of Asn119 were shown to be within a range of 2.01-2.11 A. The thermal factors for the magnesium ion in subunits are 7.08 and 4.60 A2, and the average thermal factors for the surrounding water molecules are 11.14 and 10.30 A2, respectively. The region of the nuclease subunit interactions was localized, and the alternative side chain conformations were defined for 51 amino acid residues of the nuclease dimer.

[Viscumin modulates neutrophil respiratory burst, caused by a chemotactic peptide]. / Viskumin moduliruet respiratornyi vzryv v neitrofilakh, vyzvannyi khemotaksicheskim peptidom.

The ability of viscum at different concentrations to modulate the respiratory burst in neutrophils, induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine was studied. This does not exclude the possibility that viscum can interact with the receptor of this peptide. The analysis of the primary structure of viscum revealed elements structurally analogous to the chemotactic peptide. It is assumed that viscum can exhibit the properties an antagonist of the receptor of N-formyl-methionyl-leucyl-phenylalanine, and the mechanism of action of viscum depends on its concentration.

Human recombinant laminin-binding protein: isolation, purification, and crystallization.

The mRNA of the precursor of laminin-binding protein (LBP) was isolated from a human embryo kidney cell line and cloned. The determined sequence of the LBP gene showed complete identity with the LBP genes isolated from human lung and large intestine cells. The human LBP was expressed by E. coli cells, and it was purified using Ni-NTA-Sepharose chromatography. The mobility of the homogeneous recombinant human laminin-binding protein on SDS-PAGE was 43 kD. A mixture of eight murine monoclonal antibodies, the MPLR Pool against LBP, reacted with the recombinant LBP in Western blot. The interaction of the antiidiotypical antibodies 10H10 and E6B provided evidence that the epitope binding to protein E of the tick-borne encephalitis (TBE) virus is also preserved on the human recombinant LBP. Enzyme immunoassay confirmed the ability of the recombinant LBP to interact with protein E of TBE virus. The biological activity of the recombinant LBP allowed us to perform X-ray analysis of the spatial arrangement of the LBP molecule using the recombinant protein. For this purpose, crystals of the human LBP were obtained by the standing drop version of the pore diffusion technique. The crystals appropriate for X-ray structural analysis were 0.3 x 0.1 x 0.05 mm in size. The X-ray diffraction field of the crystal extended to 2.5 A.

Atomic structure of the Serratia marcescens endonuclease at 1.1 A resolution and the enzyme reaction mechanism.

The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

[Features of the modulating effect of complexes of ribosome-inactivating proteins type II with sugars on respiratory burst of neturophils, caused by a chemotactic peptide]. / Osobennosti moduliruiushchego deistviia kompleksov ribosom-inaktiviruiushchikh belkov II tipa s sakharami na respiratornyi vzryv neitrofilov, vyzvannyi khemotaksicheskim peptidom.

It was shown that agents inducing phagocytosis (zymosan, lectins) cause changes in the number of receptors responsible for fast neutrophil reaction (chemotaxis or respiratory burst) or inhibit the binding of the agonist to its receptor. Among lectins are ribosome-inactivating proteins of type II ricin and agglutinin ricin, which penetrate the cell by binding to mannose and galactose receptors. It was shown that ribosome-inactivating proteins of type II can exhibit the properties of the antagonist of the receptor N-formylmethionylleucylphenylalanine. Ricin is more effective in modulating the respiratory burst induced by the chemotactic peptide than agglutinin ricin. The modulating effect of ribosome-inactivating proteins of type II on neutrophils is likely to be mediated by their interaction with galactose rather than mannose receptors. Presumably, the affinity of ribosome-inactivating proteins to galactose receptors increases with increasing amount of saccharides bound to the protein molecule. The modulating effect of ribosome-inactivating proteins of type II on the respiratory burst of neutrophils induced the chemotactic peptide is due to the structural peculiarities of these proteins.

Three-dimensional structure of Serratia marcescens nuclease at 1.7 A resolution and mechanism of its action.

The three-dimensional crystal structure of Serratia marcescens (Sm) nuclease has been refined at 1.7 A resolution to the R-factor of 17.3% and R-free of 22.2%. The final model consists of 3678 non-hydrogen atoms and 443 water molecules. The analysis of the secondary and the tertiary structures of the Sm nuclease suggests a topology which reveals essential inner symmetry in all the three layers forming the monomer. We propose the plausible mechanism of its action based on a concerted participation of the catalytically important amino acid residues of the enzyme active site.

[Determination of angiogenin in cow's milk based on a competitive test "pancreatic RNAase/placental inhibitor of RNAase--angiogenin"]. / Opredelenie angiogenina v korov'em moloke na osnove konkurentnoi proby "pankreaticheskaia RNKaza/platsentarnyi ingibitor RNKazy-- angiogenin".

Contents of active factor of blood vessel growth angiogenin was analyzed in cow's milk by the method of competitive test in system "pancreatic RNAase/placental inhibitor of RNAase-angiogenin". High level of RNAase in milk limits using this test. To avoid this limitation the method of RNAase elimination from milk was elaborated.

[Purification of angiogenin from cow's milk]. / Ochistka angiogenina iz korov'evo moloka.

A new method for the purification of bovine angiogenin is proposed which is based on the use of CM-cellulose, CM-Toyopearl, and Butyl-Toyopearl. Electrophoretically homogeneous protein was obtained with a 49% yield and 12,000-fold purification.

[Interaction of metal ions with the carboxyl group of Asp-5 in the active site of uridine phosphorylase from Escherichia coli K-12]. / Vzaimodeistvie ionov metallov s karboksil'noi gruppoi Asp-5 v aktivnom tsentre uridinfosforilazy iz Escherichia coli K-12.

The effects of La3+ ions on enzymatic activity and difference absorption spectra of native and fluorescein isothiocyanate (FITC) modified uridine phosphorylase from E. coli K-12 have been studied. Excess La3+, unlike Ag+, only slightly decreases the enzyme activity but provokes similar changes in the absorption spectra of both native and modified proteins. The Kd value for La3+ ions (0.2 mM) coincides with that obtained earlier for Ag+. La3+ ions (0.2 mM) have no effect on the rate of the enzyme inactivation by diethylpyrocarbonate or tetranitromethane but increases the rate of its inactivation by Woodward's reagent K (WRK). Binding of La3+ (Kd = 0.2 mM) markedly decreases the thermal stability of the enzyme which increases with a further rise in the La3+ concentration. The values of Kd (0.2 mM) as well as the difference spectra and specific interactions with WRK indicate that one of the ligands interacting with metal ions is the carboxyl group of the Asp-5 residue. According to X-ray analysis data, this residue is involved in the formation of the active center of uridine phosphorylase.

[Quantitative determination of cattle angiogenin]. / Kolichestvennoe opredelenie angiogenina byka.

Polyclonal antibodies to a milk antigen were obtained by a standard immunization procedure. The possibility was demonstrated to use the peroxidase-antibody conjugate in a competitive immunoenzymatic test. The minimal amount of the antigen determined by this method is 10 ng/ml.

Atomic structure at 2.5 A resolution of uridine phosphorylase from E. coli as refined in the monoclinic crystal lattice.

Uridine phosphorylase from E. coli (Upase) has been crystallized using vapor diffusion technique in a new monoclinic crystal form. The structure was determined by the molecular replacement method at 2.5 A resolution. The coordinates of the trigonal crystal form were used as a starting model and the refinement by the program XPLOR led to the R-factor of 18.6%. The amino acid fold of the protein was found to be the same as that in the trigonal crystals. The positions of flexible regions were refined. The conclusion about the involvement in the active site is in good agreement with the results of the biochemical experiments.

The atomic structure of Carnation Mottle Virus capsid protein.

The structure of the Carnation Mottle Virus (CMtV) capsid protein has been determined at 3.2 A resolution by the method of molecular replacement. Three-dimensional data were collected from a small number of crystals (sp.g. I23, a = 382.6 A) using the synchrotron radiation with an image plate as detector. The coordinates of Tomato Bushy Stunt Virus (TBSV) were used as a searching model. Refinement of the coordinates of 7,479 non-hydrogen atoms performed by the program XPLOR, has led to an R-factor of 18.3%. It was found that the amino acid chain fold of capsid protein is very similar to that in other icosahedral viruses. However, there are some differences in the contact regions between protein subunits and also the lack of the beta-annulus around the 3-fold icosahedral axes. The structural and biochemical results lead us to consider an alternative assembly pathway.

[Crystallization of three multiple forms of transketolase from baker's yeast]. / Kristallizatsiia trekh mnozhestvennykh form transketolazy iz pekarskikhdrozhzhei.

Three forms of baker's yeast transketolase have been revealed. These forms differed in thermal stability and elution profiles during chromatography on a phosphocellulose column and migrated with identical rates during electrophoresis in the presence of sodium dodecyl sulfate. The same forms in yeast, pig and rat liver and in different organs and tissues of the rabbit were found to be similar in their thermal stability and chromatographic properties. The relative amounts of the forms appeared to depend on the physiological state of the organism. Crystals of the three pure forms were grown using ammonium sulfate as the precipitating agent. These crystals differed morphologically and by stability upon storage. The possibility of interconversion of the transketolase forms is discussed.