Human alpha-1-proteinase inhibitor (α-1-PI) from synovial fluid has been isolated to near 90% purity. The preparation has a molecular weight near 52,000, contains 3.5 residues of methionine sulfoxide, and an amino terminal glutamine residue. Sequence studies indicate that the first 17 residues, normally present in plasma α-1-PI, are missing from this protein. The inhibitor did not form a complex with porcine pancreatic elastase but, instead, was converted to a lower molecular weight form. Sequence studies on the latter indicated that two methionyl residues, one at the P1 reactive site and the other at P8, had been oxidized. These data confirm the fact that oxidized α-1-PI may be formed , presumably by the action of myeloperoxidase. This latter effect may alter the proteinase-proteinase inhibitor balance in tissues so that excess proteolysis and abnormal tissue degradation may occur. 相似文献
Double label experiments were performed employing 13CO and either H218O or 18O2 in the presence of a CO utilizing bacterium. CO2 generated was trapped and ratios showed that the second oxygen atom in the oxidation of CO to CO2 by this bacterium comes neither from O2 nor H2O. 相似文献
Oxygen-18 exchange out of [18O]Pi catalyzed by Mg2+-activated unadenylated glutamine synthetase from was followed by 31P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the in the species P18O4, P18O316O1, P18O216O2, P18O116O3, P16O4 during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of Pi. The rate constant for exchange was 410±40 min?1 while the rate constant for net reaction (ATP formation) was 62±4 min?1. Thus exchange proceeds ~7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ( (1976) , 5558) for the Mn2+-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) , 476) was successfully used to fit the oxygen exchange data in this paper. 相似文献
The cell-free preparations from autotrophieally grown Pseudomonas saccharophila catalyzed the process of electron transport from H2 or various other organic electron donors to either O2 or NO3? with concomitant ATP generation. The respective ratios with H2 and NADH were 0.63 and 0.73, the respective ratios were 0.57 and 0.54. In contrast, the and ratios with succinate were 0.18 and 0.11, respectively. ATP formation coupled to the oxidation of ascorbate, in the absence or presence of added N,N,N′,N′-tetramethyl-p-phenylenediamine or cytochrome c, could not be detected. Various uncouplers inhibited phosphorylation with either O2 or NO3? as terminal electron acceptors without affecting the oxidation of H2 or other substrates. The NADH oxidation at the expense of O2 or NO3? reduction as well as the associated phosphorylation were inhibited by rotenone and amytal. The aerobic and anaerobic H2 oxidation and coupled ATP synthesis, on the other hand, was unaffected by the flavoprotein inhibitors as well as by the NADH trapping system. The NADH, H2, and succinate-linked electron transport to O2 or NO3? and the associated phosphorylations were sensitive, however, to antimycin A or 2-n-nonyl-4-hydroxyquino-line-N-oxide, and cyanide or azide. The data indicated that although the phosphorylation sites 1 and II were associated with NADH oxidation by O2 or NO3?, the energy conservation coupled to H2 oxidation under aerobic or anaerobic conditions appeared to involve site II only. 相似文献
The properties of several compounds useful as models for three-dimensional conformational studies and the investigation of the chemical degradation of glycopeptide linkages both of the N- and O-glycosidic type are described. Using the method of differential chemical shift in H2O and D2O as solvents, the carbon NMR spectrum of N-acetylglucosaminylasparagine, 1-N-acetyl-β-d-glucopyranosylamine, and 1-N-acetyl-2-acetamido-β-d-glucopyranosylamine has been assigned. Electron impact mass spectra of the peracetylated derivatives of the latter two compounds show a peak apparently unique to glycopyranosylamides at , no analog of which is observed in the mass spectra of other peracetylated sugars. As models of the α-O-glycosidic linkage, fully assigned carbon NMR spectra of α-methyl-N-acetylgalactosamine (GalNAc), α-methyl-3-O-methyl GalNAc,and -GlcNAc as well as the disaccharide Glc-β-1 → 3 GalNAc are reported. Because certain anomalies in the chemical shifts and 1JCH observed in the disaccharide and in O-glycosylated glycoproteins are not observed in the simple model compounds, they may result from conformational interactions in the glycopeptides. 相似文献
The interaction of tRNATyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by differential acetylation of lysine residues. The synthetase was trace-labelled in the free form and as the synthetase-tRNATyr complex with [3H]acetic anhydride. In a second step the two 3H-labelled enzyme preparations were fully acetylated with cold reagent under denaturing conditions and were mixed with synthetase that had been homogeneously labelled with excess [14C]acetic anhydride. Peptides containing labelled lysine residues were isolated after chymotryptic digestion and their ratios were determined. These ratios reflect the reactivity of primary amino groups towards acetic anhydride.Involvement of lysine side-chains in complex formation with tRNATyr was suggested from altered ratios. Out of the 22 primary amino groups of tyrosyl-tRNA synthetase at least three showed reduced reactivities towards acetic anhydride in the synthetase-tRNATyr complex by factors of 1.6, 1.9 and 6.8, respectively. The sequences around these lysine residues have been determined enabling their placement when the primary and tertiary structure of the enzyme are available (G. L. E. Koch, to be published). No lysine residue of increased reactivity in the synthetase-tRNATyr complex has been detected.Only one molecule of tRNATyr binds to the dimeric synthetase molecule under the conditions of the differential labelling. If the binding site for the tRNA is on one of the two identical subunits, any observed decrease in chemical reactivity of a particular lysine residue should not exceed a factor of two. The detection of a lysine residue which reacts about seven times more slowly in the synthetase-tRNA complex could therefore indicate that the single binding site is formed by both enzyme subunits. 相似文献
Mass spectral analyses of the CO2 liberated in the luciferin-luciferase and firefly luciferin-luciferase reactions run in the presence of 17O2 and H218O show that the product is predominantly C18O16O (mass 46) and not C17O16O (mass 45). Incorporation of 18O into medium CO2 by exchange does not account for the observed results. These experiments provide evidence that the and firefly bioluminescence reactions proceed via a linear peroxide mechanism rather than the dioxetane mechanism and suggest that a common mechanism may underly many bioluminescence reactions. 相似文献
Complexes of the formula cis-[Pt(H~N)(L)Cl2], where (H~N) are the protonated diamines including 3-aminoquinuclidine, N-aminopiperidine, piperazine, N-methylpiperazine, 1,1,4-trimethylpiperazine, and N-methyl-1,4-diazabicyclo [2,2,2] octane (N-methyl-dabco) and L = SCN?, NO2?, Br?, and F?, were synthesized from the protonated diamine complexes, [Pt(H~N)Cl3]. The antitumor activities of the complexes were evaluated in vitro against L1210 murine leukemia cells, and ID50 values for the L-substituted complexes were compared to values of the parent complexes. In each case it was found that replacement of a chloride ion by SCN?, NO2?, Br?, or F?, either reduced or completely eliminated antitumor activity. This effect is explained in terms of the trans-directing ability of the ligand, L, compared to chloride. The NO2-substituted complex of 3- aminoquinuclidine was tested in vivo and found to exhibit little or no antitumor activity. 相似文献
Structural studies of homozygous glycophorin AM were undertaken by monitoring the 13C methyl resonances of 13C reductively methylated glycophorin AM (contains five Lys residues, and the N-terminal Ser residues) in various forms of glycosylation. The results indicate that removal of the α-d-NeuAc residues does not affect the structure about the N-terminal Ser residue. However, removal of the fifteen O-linked oligosaccharide units results in a structural effect about the N-terminal Ser residue. Partial methylation experiments performed on native glycophorin AM and deglycosylated glycophorin AM indicate that methylation of the lysine residue(s) may influence the structure about the N-terminal Ser residue, especially in the case of deglycosylated AM. 相似文献
The relative effectiveness of oxidizing (.OH, H2O2), ambivalent (O2?) and reducing free radicals (e? and CO2?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ((enz)) were measured and the rates of damage to membranes ((mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. .OH, O2? and H2 O2 were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e? and CO2? were the least effective. (enz) values of O2? and H2O2 were 10-times and of .OH 15-times that of e?. (mb) values were quite similar for e? and H2O2 (about twice that of O2?), while that of .OH was 3-times that of O2?. Hence, with respect to (mb): , and with respect to (enz): . The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H2O2 added as a chemical reagent and H2O2 formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H2O2 but markedly susceptible to the latter. 相似文献
NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100–250 μequiv · h?1 · mg?1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 → Photosystem II → dimethylquinone reaction supports phosphorylation with a ratio of 0.25–0.35 and proton uptake with values of 0.67 (pH 8)–0.85 (pH 6). These are close to the value of 0.3–0.38 and the values of 0.7–0.93 found in parallel experiments for the H2O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I?, ferrocyanide). 相似文献
[4-14C]Cholesterol was incubated with an adrenocortical preparation in the presence of 16O2 and 18O2 devoid of significant 16O18O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of 289, 291, and 293 (comprising the C8 C-20 to C-27 side-chain and containing, respectively, 16O2, 16O18O, and 18O2) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of 291 would have been absent. 相似文献
α-(1→2)--Fucosidase, β--galactosidase and galactose oxidase are sterically hindered by certain types of branching in the oligosaccharide chains. 1) β--Galactosidase will not cleave galactose when the penultimate sugar carries a sialic acid residue as in I. 2) Galactose Oxidase will not oxidize the galactose residue in trisaccharide I but will in II. Moreover, neither galactose nor -acetylgalactosamine, glycosidically bound as in III, is susceptible to oxidation with galactose oxidase until the α-(1→2) linkage between them is cleaved by . 3) α-(1→2)--Fucosidase action is inhibited by or galactosyl residue, as in III and IV. Removal of the terminal sugars makes the fucosyl residue susceptible to fucosidase action. 相似文献
The stoichiometry of free NADPH oxidation in phenobarbital induced rabbit liver microsomes was measured by means of registering the rates of NADPH, H+ and O2 consumption and and H2O2 production. ratio is approximately I indicating that about half H2O2 results from dismutation, the second half being formed directly. ΔNADPH:ΔH2O2 and ΔO2:ΔH2O2 ratios exceed I and therefore another product of the reaction is water. The fact that the ratio (ΔNADPH-ΔH2O2):(ΔO2-ΔH2O2) is 2 allows one to consider direct 4-electron O2 reduction as the major way of water formation rather than endogenous substrate hydroxylation. 相似文献
Transamination of erythro-β-hydroxy-l-aspartate catalyzed by pig heart aspartate aminotransferase (EC 2.6.1.1) was studied with both normal and α-deuterated substrate in . The overall transamination reaction, with α-ketoglutarate as amino group acceptor, showed no primary substrate isotope effect. However, one of the elementary reactions between two enzyme-substrate complexes was found to exhibit large primary isotope effects in both the forward and the reverse directions. This same reaction also showed a twofold solvent isotope effect in the reverse direction, but D2O had only a negligible effect in the forward direction. These data were interpreted to indicate that the substrate α-hydrogen arises from a Bronsted acid with two equivalent hydrogens. Another elementary reaction, which is 100-fold slower, was also studied since it appeared to be one of the principal rate-determining steps in the overall reaction. This step was not affected by substrate deuteration but exhibited large solvent isotope effects in both directions. 相似文献
Two similar but distinct forms of α1-protease inhibitor (α1-PI) have been isolated and purified 120-fold to homogeneity from the plasma of female, white Swiss (Ha/ICR) mice. The two inhibitors can be separated by chromatography on DEAE-cellulose using a shallow NaCl gradient at pH 8.9 for elution. Because of their differing specificities for elastase and trypsin we have labeled the two inhibitors α1-PI(E) and α1-PI(T), respectively. The apparent Mr for both proteins, as estimated by gel exclusion chromatography, is approximately 53,000 daltons. However by polyacrylamide gel electrophoresis in the presence of SDS, α1-PI(T) has an apparent mr of 65,000 while the apparent mr of α1-PI(E) is 55,000. These results suggest differences in charge and carbohydrate composition. The two mouse inhibitors also have different AT-terminal amino acids. Like human α1-PI the mouse inhibitors form stable complexes with proteases. However they differed from human α1-PI in that they were not found to neutralize either human thrombin or plasmin. While α1-PI(E) inhibits bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase, α1-PI(T) is an effective inhibitor only of trypsin. Plasma levels of α1-PI(E) increase significantly 24 h after stimulation of the acute phase reaction while those of α1-PI(T) do not. Our data suggest that α1-PI(E) and α1-PI(T) are products of different genes. 相似文献
Superoxide dismutase and catalase were not detected in . and several other species of some of which consume oxygen and secrete H2O2. . in suspension formed O2? in the presence of NADH and flavins and extracts of . formed O2? in the presence of either NADH or NADPH. The lack of superoxide dismutase in . could not be attributed to superoxide dismutase in the complex medium in which the organisms were grown because organisms grown in medium in which the superoxide dismutase had been inactivated by heat still contained undetectable amounts. Mycoplasmas appear to be an exception to the rule that organisms which consume O2 synthesize superoxide dismutase. 相似文献
The complexes trans-[ReL2(dppe)2]BF4 (I, L = CO, CNR with R = Me,But, C6H4OMe-4, C6H4Me-4, C6H4Cl-4 and C6H3Cl2-2,6) were prepared from the reaction of the parent dinitrogen complex trans-[ReCl(N2)(dppe)2] with the appropriate ligand, in thf and in the presence of TlBF4.Their redox properties were studied by cyclic voltammetry at a Pt electrode in [Bu4N][BF4]/thf or acetonitrile; they undergo a one-electron reversible oxidation and the observed EOX values were applied to test the validity of a proposed general expression —derived from the electrochemical ligand (PL) and 16-electron metal site (Es, β) parameters [6]— to estimate EOX for 18-electron octahedral complexes of the type [M′sL2] with a square planar 14-electron metal centre {M′s}. Es and β parameters were also estimated for the auxilliary {ReL(dppe)2}+ (L = CO or CNR) centres. 相似文献
(22)-[22-3H1]-Cholesterol was incubated with an adrenocortical preparation and the isolated (22)-[22-3H1]-22-hydroxycholesterol had a small loss of radioactivity, proving that direct replacement of the hydrogen from the now hydroxylated position occurred.In addition [1-3H1]-4-methylpentanol was isolated, which also had incurred a relatively small loss of its specific activity, thereby excluding (20)-3β,20-dihydroxycholest-5-en-22-one as an important metabolite in the degradation of cholesterol to pregnenolone by adrenal tissue. 相似文献