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81.
T. M. Sossong Jr. Sergei V. Khangulov R. C. Cavalli Dianne Robert Soprano G. C. Dismukes D. E. Ash 《Journal of biological inorganic chemistry》1997,2(4):433-443
Rat liver arginase contains a dinuclear Mn2(II,II) center in each subunit having EPR properties similar to those observed in Mn-catalases. The principal physiologic
role of arginase is catalyzing the hydrolytic cleavage of l-arginine to produce l-ornithine and urea. Here we demonstrate that arginase catalyzes the disproportionation of hydrogen peroxide by a redox mechanism
analogous to Mn-catalases, but at rates that are 10–5 to 10–6 of k
cat for the Mn-catalases, and also exhibits peroxidase activity. The dinuclear Mn2(II,II) center is essential for maximal catalase activity, since both the H101N and H126N mutant arginases containing only
one Mn(II)/subunit have catalase activities that are <3% of that for the wild-type enzyme. Like the Mn-catalases, the catalase
activity of arginase is not inhibited by millimolar concentrations of CN–, the most potent inhibitor of heme catalases, or by EDTA, a chelator of free metal ions. The catalase activity of arginase
is not significantly inhibited by Cl– or F–, in contrast to Mn-catalases, while potent inhibitors of the hydrolytic activity are also effective inhibitors of the catalase
activity. These results suggest that lower affinity of hydrogen peroxide to the active site of arginase contributes to the
lower catalase activity. EPR spectroscopy reveals that potent inhibitors of the hydrolytic reaction, including N
ω-hydroxy-l-arginine, l-lysine, and l-valine, decouple the electronic interaction between the Mn2+ ions, most probably by removing a μ-bridging ligand or by increasing the intermanganese separation. The capacity for arginase
to deliver a hydroxide ion to hydrolyze the l-arginine substrate is suggested to arise from a "dinuclear effect", wherein the two metal ions contribute more or less equivalently
in deprotonation of metal-bound water molecule. Structure-reactivity analyses of these reactions will provide insights into
the factors that control redox versus hydrolytic function in dimanganese clusters.
Received: 18 November 1996 / Accepted: 7 April 1997 相似文献
82.
83.
Density changes in herpesvirus-infected cells 总被引:1,自引:0,他引:1
84.
Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells. 总被引:10,自引:3,他引:7
J R Emanuel S Garetz J Schneider J F Ash E J Benz Jr R Levenson 《Molecular and cellular biology》1986,6(7):2476-2481
We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype. 相似文献
85.
Activation of volatile fatty acids in bovine liver and rumen epithelium. Evidence for control by autoregulation 总被引:3,自引:2,他引:1 下载免费PDF全文
1. The total capacities of homogenates of bovine liver and rumen epithelium to activate acetate, propionate and butyrate were determined. 2. Activating capacities were assayed by measuring the rate of formation of the corresponding CoA esters. The methods used for determining the concentrations of the CoA esters allowed the CoA esters of acetate, propionate and butyrate to be distinguished. It was thus possible to investigate the effect of the presence of a second volatile fatty acid on the rate at which a given volatile fatty acid was activated. 3. The propionate-activating capacity in rumen epithelium was decreased by about 87% in the presence of butyrate, the acetate-activating capacity in liver was decreased by about 55% in the presence of either propionate or butyrate, and the butyrate-activating capacity in liver was decreased by about 40-50% in the presence of propionate. 4. All three activating capacities in liver appeared to be located in the mitochondrial matrix and membrane. The three activating capacities had similar locations to each other in rumen epithelium as well, although in this case activity was more evenly divided between the mitochondria and the cytoplasm. 5. The relative activating capacities towards the volatile fatty acids in the two tissues, together with the ability of one volatile fatty acid to inhibit the activation of another volatile fatty acid, appear to ensure that butyrate is mainly metabolized in the rumen epithelium and that propionate is metabolized in the liver. 相似文献
86.
Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16S rRNA 总被引:12,自引:0,他引:12
87.
D E Ash N J Papadopoulos G Colombo J J Villafranca 《The Journal of biological chemistry》1984,259(6):3395-3398
The question of the stoichiometry of copper bound to dopamine beta-hydroxylase and the number of copper atoms required for maximal activity was addressed in this study. Incubation of tetrameric enzyme from bovine adrenal medulla with 64Cu2+ followed by rapid gel filtration yielded an enzyme containing 8.3-8.9 mol of Cu/mol of tetramer. An identical stoichiometry was obtained by analysis of bound copper by atomic absorption methods. NMR and EPR were used to monitor titrations of the enzyme with Cu2+ and showed that the longitudinal relaxation rate of solvent water protons and the amplitude of the signal at g approximately 2 increased linearly up to a copper to protein ratio of approximately 8. Additional titrations also indicate that an enzyme-Cu2+-tyramine-CN- inhibitory complex was formed when 8 mol of Cu2+ are bound per mol of enzyme. The rate of inactivation of dopamine beta-hydroxylase by the mechanism-based inhibitor 2-Br-3-(p-hydroxyphenyl)-1-propene was measured and used as a method to follow enzymatic catalysis. An increase in rate was observed with increasing Cu2+ up to a protein to Cu2+ ratio of 8 Cu/tetramer. The rate becomes constant after this ratio is achieved. These data indicate that dopamine beta-hydroxylase specifically binds 8 mol of Cu/tetramer and that this stoichiometry is required for maximal activity. 相似文献
88.
Eggshells of Nematodirus battus leaked trehalose 4 hr after being stimulated to hatch, and became permeable to trypan blue at their poles; 80% of eggs were stained blue 24 hr later. Exogenous application of ruthenium red significantly inhibited chill- and sodium fluoride-stimulated hatching, 50% hatch inhibition occurring in 44.67 ± 2.2 and 8.5 ± 1.5 μM, respectively. Lanthanum chloride, however, was not as inhibitory as ruthenium red on fluoride-stimulated hatching, 50% occurring at 31.60 ± 1.25 μM. A Scatchard plot of the competitive binding of ruthenium red to eggshells demonstrated a high-affinity binding site for calcium, KCa′ = 1.92 μM and a second, low-affinity site, KCa′ = 1169.60 μM. Ruthenium red binding was significantly reduced by several enzymes, e.g., EGTA-buffered trypsin reduced binding by 73%. Radioiodinated concanavalin A also bound competitively to the eggshells in the presence of α-d-glucosyl-α-d-glucopyranoside and α-methyl-d-mannopyranoside. Eggshells incorporated phosphorus-32 from ATP after chilling or on exposure to sodium fluoride; gel filtration of solubilized homogenates of these samples showed that two proteins were radiolabelled with molecular weights of 38 × 103 and 8 × 103 Da, respectively. This phosphorylation was inhibited by N-ethylmaleimide, which also prevented hatching. 相似文献
89.