The carbohydrate moiety of alpha-galactosidase from Trichoderma reesei.

Alpha-galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by beta-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: alpha1-6 D-mannopyranosyl-alpha-D-glycopyranoside and alpha1-6 D-glucopyranosyl-alpha-D-galactopyranoside and one trimer: alpha-D-glucopyranosyl-alpha1-2 D-mannopyranosyl-alpha1-6 D-galactopyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo-beta-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo-beta-N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of alpha-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of alpha-galactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described.

Role of methionine in the active site of alpha-galactosidase from Trichoderma reesei.

alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH. Galactose (a competitive inhibitor of alpha-galactosidase; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously, a methionine located in the active site of alpha-galactosidase is more accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide.

[Biologically active substances during treatment of pulmonary hypertension with ATP infusions immediately after general anesthesia and surgery of hypervolemic congenital heart defects]. / Biologicheski aktivnye veshchestva v usloviiakh terapii legochnoi gipertenzii infuziiami ATF v blizhaishem periode posle obshchei anestezii i likvidatsii gipervolemicheskikh vrozhdennykh porokov serdtsa.

In 95 patients with hypervolemic congenital heart valve defects (42 with interventricular and interatrial septal defects, and 53 with patent ductus arteriosus) mixed venous blood levels of serotonin, histamine, adrenalin, noradrenaline, total 11-hydroxycorticosteroids were studied using spectrofluorimetry, cyclic adenosine- and guanosine monophosphate (cAMP and cGMP), prostaglandins E + A and F2 alpha and their interaction were assessed using radioimmunoassay and correlation analysis, respectively. Immediately after general anesthesia and surgical correction of septal defects using cardiopulmonary bypass adrenalin level was increased, while noradrenaline, cAMP, cGMP, prostaglandin E + A levels remained high. Similar pattern was observed in patients after arterial duct ligation. Mean pulmonary artery pressure was elevated in both groups of patients (above 4 kPa). Effective therapy of pulmonary hypertension with intravenous ATP infusions immediately after general anesthesia and elimination of septal defects (20 patients) was accompanied by a decline in noradrenaline, cGMP and total 11-hydroxycorticosteroid level (with adrenalin level increased), while after open arterial duct ligation (20 patients) it was followed by a drop in noradrenaline and prostaglandin F2 alpha level (with adrenalin level increased) and the activation of the interaction of biologically active substances aimed predominantly at attenuation of vasoconstrictor reactions in the lungs.

[Biologically active substances in patients with hypervolemic congenital heart defects undergoing general multicomponent anesthesia]. / Biologicheski aktivnye veshchestva u bol'nykh s gipervolemicheskimi vrozhdennymi porokami serdtsa v usloviiakh obshchei mnogokomponentnoi anestezii.

In 95 patients with hypervolemic congenital heart diseases (42 with interventricular and interatrial septal defects, and 53 with patent ductus arteriosus) mixed venous blood levels of serotonin, histamine, adrenalin, noradrenaline, total 11-hydroxycorticosteroids were studied using spectrofluorimetry, cyclic adenosine- and guanosine-monophosphate (cAMP and cGMP), prostaglandins E+A and F2 alpha and their interaction were assessed using radioimmunoassay and correlation analysis, respectively. Before surgical treatment the patients showed higher (than in the control) concentrations of histamine, adrenalin, noradrenaline, cAMP, and cGMP, prostaglandins E+A. During anesthesia including fentanyl, morphine, diazepam, N2O:O2 in patients with heart valve defects cGMP level was increased and in patients with patent ductus arteriosus cAMP level was increased during analogous anesthesia with morphine replaced by halothane.

[Immunological disorders of homeostasis and the methods of their correction for the programmed and preoperative treatment of patients with nonspecific aorto-arteritis]. / Immunologicheskie narusheniia gomeostaza i metody ikh korrektsii dlia programmnogo i predoperatsionnogo lecheniia bol'nylh nespestsificheskim aorto-arteriitom.

The authors examined 29 patients with aorto-arteritis of unspecific origin of different duration and variants of affections of the major vessels. The patients were divided into groups with active and inactive course of the inflammatory process. The changes of immunological values and the phagocytosis data were more marked in the group with the active phase of the disease. These changes correlated with the data on unspecific inflammation and the clinical picture. Twenty-two patients were treated by hemosorption and immunocorrective measures. The fibrinogen concentration reduced, the ESR was normalized, and C-reactive protein became negative. The concentration of circulating immune complex and immunoglobulins diminished, the phagocytosis values became close to normal. It is concluded that hemosorption is an effective method of correction of immunological disorders in unspecific aorto-arteritis, both as an independent therapeutic method and as a measure of preoperative management.

[Determination of risk factors for tuberculosis in children having contact with tuberculosis patients]. / Opredelenie riska zabolevaniia tuberculezom detei, kontakti ruiushchikh s bol'nymi tuberkulezom.

Analysis is given of the specific and nonspecific features in 100 tuberculosis patients and 500 children aged under 7 years having contact with tuberculosis patients. A complex of factors with a high prognostic value was defined from the set of the studied features and a scale for determining the tuberculosis risk in children was constructed. Estimating the risk degree by a prognostic table, allows one to define children at a high and maximal infection risk for the individual choice of preventive measures.

[Distribution of the tempo-dichlorotriazine spin label in the immunoglobulin molecule]. / Raspredelenie spinovoi metki tempo-dikhlorotriazina po molekule immunoglobulina.

Spin-label 14C-TEMPO-dichlorotriazine distribution in Fab- and Fc-fragments of monoclonal myeloma immunoglobulin IgG (lambda) 1 Bel, in their heavy and light chains, and tryptic fragments of chains was investigated. Radio-active spin label in proteolytic Fab- and Fc-fragments, in heavy and light chains, and in separate peptides was detected. 3 cm and 2 mm band ESR spectra of spin-labeled IgG in water solutions was studied. It was shown that the models of highly anisotropic motion, isotropic motion and slow isotropic motion of spin label around globule or the description of experimental ESR spectra of TEMPO-dichlorotriazine spin-labeled immunoglobulins do not work.

[Analysis of existing models of spin label movement during spin labeling of biological macromolecules]. / Analiz sushchestvuiushchikh modelei dvizheniia spinovoi metki pri spin-mechenii biologicheskikh makromolekul.

The spin-labeled bovine serum albumin and IgG were studied in search of an experimental approach for comparison of different models of rotational mobility of spin label. These models are: the model of isotropic motion of spin label together with the macromolecule (IM); the model of highly anisotropic motion of spin label (HAM); and the model of slow isotropic motion of label around the binding site (SIML). The experimental spectra were measured on a common X-band ESR spectrometer and on the unique 140 GHZ (lambda = 2 mm) ESR spectrometer under the same conditions. Theoretical spectra were computer-calculated according to Freed's theory. We have found, that the results of temperature-viscosity experiments in X-band are contradictory to the model of IM both for the BSA and IgG species. The models of HAM and SIML for the BSA give identical X-band spectra. The bovine serum albumin spectra in the 2 mm region strongly contradict to the assumptions of the HAM model. Also, the SIML model fails to describe the experimental spectra in terms of isotropic motion of the spin label around the binding site. X-band spectra of IgG can not be explained by the SIML model, while the same spectra in the 2 mm region can not be explained by the HAM model.

[Spectral division of conformational states of spin-labeled tRNA Phe from Escherichia coli]. / Spektral'noe razdelenie konformatsionnykh sostoianii na spin-mechenoi tRNK Phe iz Escherichia coli.

A spectral division method of two conformational states of spin-labeled macromolecules is presented. The method is suitable in conditions of highly anisotropic motion of spin label and is based on titration of experimental spectra of spin-labeled macromolecule by theoretical ones. Theoretical spectra simulation uses the Freed theory and spin-Hamiltonian parameters, derived from independent experiments. Nomogrammes and formula for calculation of order parameter Sz and correlation time tau c in temperature-viscosity experiment are available. The method was applied to spectral division of two conformational states of spin-labeled tRNAPhe from E. coli and spectral parameters Sz and tau c were obtained for both states. ESR spectra of these conformational states at t degree = 20 degrees differ strongly from one another by order parameter Sz. The first conformer, that is characterised by a greater order parameter has no globular conformational transition (in terms of changes of the hydrodynamic macromolecule radius) between 2 degrees and 20 degrees, but local conformational changes take place in this temperature region.

[Study on the conformational state of Escherichia coli tRNA-Phe in solution by ESR-spectometry without modulation]. / Izuchenie konformatsionnoi situatsii v vodnykh rastvorakh spin-mechenoi tRNA-Phe iz Escherichia coli bezomduliatsionnym metodom EPR.

ESR-spectrometry without modulation of the magnetic field was used for registering the EST spectral line shape (with shape distortion about 0.1 percent) of spin-labeled Escherichia coli tRNAPhe. The analysis of line shape of two different spin-labels in position 8 (S4U) revealed that tRNAPhe in solution always exists as a mixture of at least two conformers, the equilibria between conformers being dependent on pH, concentration of magnesium and the biological state of tRNA (deacylated, aminoacyl- or peptidyl-tRNA). There are no large structural rearrangements upon aminoacylation or peptidylation of tRNA, the observed small changes of spectral line shape are due to the changes in conformational equilibria.

Study on conformational states of Escherichia coli tRNAPhe in solution by a modulation-free ESR-spectrometer.

A modulation free Electron Spin Resonance spectrometer was used for the registration of spectral absorption lines of a spin-labeled Escherichia coli phenylalanine tRNA in solution with low (less than 0.1%) line shape distortion. The analysis of line shape of two different spin-labels introduced into position 8 revealed that phenylalanine tRNA in solution exists as a mixture of two conformers, the equilibria between conformers being dependent on pH, concentration of magnesium and functional state of tRNA (deacylated, aminoacylated or peptidylated). There are no overall structural rearrangements upon aminoacylation or peptidylation of tRNA. The observed small changes of spectral line shape can be assigned to shifts in conformational equilibria.

[Free-radicals formation in reductions of flavin. Study on the reduction of aminoethylcellulose-bound flavin]. / Svobodnye radikaly v reaktsii vosstanovleniia flavina. Nzuchenie Vosstanovleniia flavina, prishitogo na aminoetiltselliulozy

The reduction of flavin by reduced diphosphopyridine nucleotide and photoreduction were studied spectrophotometrically. Flavin was covalently bound to aminoethylcellulose, therefore the interaction between flavin molecules was excluded. Nevertheless a considerable quantity of free radicals was demonstrated under these conditions. The experimental dependence of the radical concentration as a function of reduction degree is readily explained if the reduction of flavin proceeds in two consecutive one-electron steps. The ratio of the rate constants of both reactions for the reduction of flavin by NADH k2'/k1' was equal to 4, for the photoreductions k2'/k1' to 6.5.