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M J Barber

Publications and source records attributed to M J Barber.

At least 73 records · Page 4Linked to original sources
PubMed1990

Spinach Nitrate Reductase : Effects of Ionic Strength and pH on the Full and Partial Enzyme Activities.

Initial velocity studies of immunopurified spinach nitrate reductase have been performed under conditions of controlled ionic strength and pH and in the absence of chloride ions. Increased ionic strength stimulated NADH:ferricyanide reductase and reduced flavin:nitrate reductase activities and inhibited NADH:nitrate reductase, NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities. NADH:dichlorophenolindophenol reductase activity was unaffected by changes in ionic strength. All of the partial activities, expressed in terms of micromole 2 electron transferred per minute per nanomole heme, were faster than the overall full, NADH:nitrate reductase activity indicating that none of the partial activities included the rate limiting step in electron transfer from NADH to nitrate. The pH optimum for NADH:nitrate reductase activity was determined to be 7 while values for the various partial activities ranged from 6.5 to 7.5. Chlorate, bromate, and iodate were determined to be alternate electron acceptors for the reduced enzyme. These results indicate that unlike the enzyme from Chlorella vulgaris, intramolecular electron transfer between reduced heme and Mo is not rate limiting for spinach nitrate reductase.

Journal Article↗
PubMed1989

Conserved domains in molybdenum hydroxylases. The amino acid sequence of chicken hepatic sulfite oxidase.

The amino acid sequence of the molybdenum-containing domain of chicken hepatic sulfite oxidase has been determined by Edman degradation of the purified protein. Combining these data with those previously published for the heme-containing domain (Guiard, B., and Lederer, F. (1979) Eur. J. Biochem. 100, 441-453) indicates that each subunit of the homodimer comprises a single polypeptide chain containing 460 amino acid residues (Mr = 50,545). Comparison of the sequence with the cDNA-deduced sequence of assimilatory nitrate reductase from Arabidopsis thaliana shows a substantial degree of sequence conservation in the regions of the proteins that have been identified as comprising the Mo-pterin- and cytochrome b557-binding domains. These results suggest that the sequences forming the molybdenum-binding domains of the molybdenum hydroxylases may have evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1989

Oxidation--reduction midpoint potentials of the flavin, haem and Mo-pterin centres in spinach (Spinacia oleracea L.) nitrate reductase.

Oxidation-reduction midpoint potentials have been determined for the flavin, cytochrome b557 and Mo-pterin prosthetic groups of spinach (Spinacia oleracea L.) assimilatory nitrate reductase using visible, c.d. and room-temperature e.p.r. potentiometric titrations. At pH 7 and 25 degrees C, the midpoint potential for the FAD/FADH2 couple was determined by c.d. potentiometry to be -280 +/- 10 mV (n = 2). The redox potential for reduction of the haem was determined by visible potentiometry to be -123 +/- 10 mV (n = 1), significantly lower than the previously published value of -60 mV [Fido, Hewitt, Notton, Jones & Nasrulhaq-Boyce (1979) FEBS Lett. 99, 180-182]. Potentials for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) redox couples, determined by room-temperature e.p.r. potentiometry, were found to be +2 +/- 20 and -6 +/- 20 mV respectively. These values are very similar to the values previously determined for the FAD, haem and Mo-pterin centres in assimilatory nitrate reductase isolated from the unicellular green alga Chlorella vulgaris and indicate a close thermodynamic similarity between the two enzymes.

Bacterial Proteins↗
PubMed1989

Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase.

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.

Amino Acid Sequence↗
PubMed1989

EPR and kinetic analysis of the interaction of halides and phosphate with nitrate reductase.

Electron paramagnetic resonance spectra obtained during turnover of the Mo center of NADH:nitrate reductase at pH 8 were comprised of two Mo(V) species, signal A (g1 = 1.996, g2 = 1.969, g3 = 1.967, A1H = 1.25 mT, A2H = 1.18 mT, and A3H = 1.63 mT) and signal B (g1 = 1.996, g2 = 1.969, and g3 = 1.967), the former exhibiting superhyperfine interaction due to strong coupling with a single, exchangeable proton. Binding of halides and nitrite to the Mo center increased the proportion of signal A whereas phosphate had no effect on the EPR line shape. Halides decreased and phosphate increased the rates of enzyme activities involving the Mo center (NADH:nitrate reductase and reduced methyl viologen:nitrate reductase), but neither had any effect on activities involving FAD (NADH:ferricyanide reductase) or heme (NADH:cytochrome c reductase), indicating specific binding of halides to the Mo center. Halides were found to be weak, mixed competitive-noncompetitive inhibitors (Cl- KI = 39 mM, mu = 0.2 M, pH 8) of nitrate reductase forming a catalytically inactive ternary halide-nitrate-enzyme complex. Inhibition patterns changed from nearly noncompetitive (F-) to nearly competitive (I-). The weakening of nitrate binding due to halide binding correlated with increased halide electronegativity rather than ionic radius. In contrast, phosphate (Kd = 7.4 mM, mu = 0.2 M, pH 8) and arsenate were determined to be nonessential activators, characterized by a constant value of (Vmax/Km)app, increasing nitrate reductase activity by weakening nitrate binding without affecting the stability of the transition state. Phosphate had no effect on product inhibition by nitrite (KI = 0.33 mM) or the oxidation-reduction midpoint potentials of the Mo center.

Arsenates↗
PubMed1989

Chloride inhibition of spinach nitrate reductase.

Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V(max) and K(m) for NO(3) (-) yielding V(max) values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K(m) values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K(m) of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a K(i) of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO(3) (-) complex. Binding of Cl(-) to the enzyme-NO(3) (-) complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase.

Journal Article↗
PubMed1988

Microcoulometric analysis of trimethylamine dehydrogenase.

Trimethylamine dehydrogenase, which contains one covalently bound 6-S-cysteinyl-FMN and one Fe4S4 cluster per subunit of molecular mass 83,000 Da, was purified to homogeneity from the methylotrophic bacterium W3A1. Microcoulometry at pH 7 in 50 mM-Mops buffer containing 0.1 mM-EDTA and 0.1 M-KCl revealed that the native enzyme required the addition of 3 reducing equivalents per subunit for complete reduction. In contrast, under identical conditions the phenylhydrazine-inhibited enzyme required the addition of 0.9 reducing equivalent per subunit with a midpoint potential of +110 mV. Least-squares analysis of the microcoulometric data obtained for the native enzyme, assuming uptake of 1 electron by Fe4S4 and 2 electrons by FMN, indicated midpoint potentials of +44 mV and +36 mV for the FMN/FMN.- and FMN.-/FMNH2 couples respectively and +102 mV for reduction of the Fe4S4 cluster.

Ferrous Compounds↗
PubMed1988

Circular dichroism and potentiometry of FAD, heme and Mo-pterin prosthetic groups of assimilatory nitrate reductase.

Oxidation-reduction midpoint potentials for flavin, heme, and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase (NR) from Chlorella vulgaris were measured at room temperature by using CD and EPR potentiometry. The CD changes accompanying reduction of each prosthetic group were determined by using enzyme fragments containing either FAD or heme and molybdenum prosthetic groups, obtained by limited proteolysis, and by poising the enzyme at various redox potentials in the presence of dye mediators. Limited proteolysis did not appear to alter the environment of the prosthetic groups, as judged by their CD spectra. Also, CD potentiometric titration of FAD in intact NR (Em' = -272 mV, n = 2) gave a similar value (Em' = -286 mV) to the FAD of the flavin-containing proteolytic domain, determined by visible spectroscopy. Less than 1% of the flavin semiquinone was detected by EPR spectroscopy, indicating that Em' (FAD/FAD.-) may be more than 200 mV lower than Em' (FAD.-/FADH-). Reduction of heme resulted in splitting of both Soret and alpha CD bands into couplets. The heme Em' was -162 mV (n = 1) determined by both CD and visible spectroscopy. Reduction of Mo-pterin was followed by CD at 333 nm, and Mo(V) was monitored by room temperature EPR spectroscopy. Most of the change in the Mo-pterin CD spectrum was due to the Mo(VI)/Mo(V) transition. The Em' values determined for Mo(VI)/Mo(V) were +26 mV by CD and +16 mV by EPR, whereas Mo(V)/Mo(IV) values were -40 mV by CD and -26 mV by EPR.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1988

Detection of phagocyte-derived free radicals with spin trapping techniques: effect of temperature and cellular metabolism.

Human neutrophils activated with either particulate or soluble stimuli generate oxygen-centered free radicals which are detected by spin trapping in conjunction with electron spin resonance (ESR) spectroscopy. We investigated the effect of temperature on ESR spectra resulting from stimulation of human neutrophils with phorbol myristate acetate (PMA) or opsonized zymosan in the presence of the spin trap, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO). At 20 degrees C with either stimuli, neutrophil superoxide production was manifested predominantly as the superoxide spin-trapped adduct, 5,5-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy (DMPO-OOH). In contrast, at 37 degrees C, the hydroxyl spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH) was dominant. No evidence of hydroxyl radical (defined as the methyl spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy, DMPO-CH3) was observed, suggesting that elevated temperatures increased the rate of DMPO-OOH conversion to DMPO-OH. In addition, the elevated temperature activated a neutrophil reductase which accelerated the rate of DMPO-OH reduction to its corresponding hydroxylamine, 2,2-dimethyl-5-hydroxy-1-hydroxypyrrolidine. This bioreduction was dependent upon the presence of both superoxide and a phagocyte-derived factor (possibly a thiol) released into the surrounding media.

Cyclic N-Oxides↗
PubMed1988

Stoichiometry of electron uptake and oxidation-reduction midpoint potentials of NADH:nitrate reductase.

Microcoulometric titrations of NADH:nitrate reductase at 25 degrees C in Mops buffer, pH 7.0, showed that the native enzyme, containing functional FAD, haem and Mo, required addition of five electrons for complete reduction. Reduction of the native enzyme occurred in three waves corresponding to addition of reducing equivalents to the centres in the order: Mo, haem, FAD. Oxidation-reduction midpoint potentials (E'0) for the various redox couples were calculated to be as follows: MoVI/MoV, +16 mV; MoV/MoIV, -27 mV; haemoxidized/haemreduced, -172 mV; FAD/FADH2, -283 mV. The values for the haem and flavin are in excellent agreement with those obtained by visible titrations, namely -164 mV and -288 mV respectively. In contrast, the results for the Mo centre are 28-50 mV more positive than the values previously determined by e.p.r. analysis of frozen enzyme samples poised at defined potentials at 25 degrees C and suggest different pH-dependencies or entropies of reduction for the Mo couples.

Chlorella↗
PubMed1987

EPR properties of the Ni-Fe-C center in an enzyme complex with carbon monoxide dehydrogenase activity from acetate-grown Methanosarcina thermophila. Evidence that acetyl-CoA is a physiological substrate.

The carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila was further studied by EPR spectroscopy. The as purified enzyme exhibited no paramagnetic species at 113 K; however, enzyme reduced with CO exhibited a complex EPR spectrum comprised of two paramagnetic species with g values of g1 = 2.089, g2 = 2.078, and g3 = 2.030 (signal I) and g1 = 2.057, g2 = 2.049, and g3 = 2.027 (signal II). Isotopic substitution with 61Ni, 57Fe, or 13CO resulted in broadening of the EPR spectra indicating a Ni-Fe-C spin-coupled complex. Pure signal II was obtained following treatment of the CO-reduced enzyme with acetyl-CoA but not by addition of acetyl phosphate or CoASH. Acetate-grown cells were highly enriched in acetate kinase (EC 2.7.2.1) and CoASH-dependent phosphotransacetylase (EC 2.3.1.8) activities. These results suggest acetyl-CoA is a physiological substrate for the carbon monoxide dehydrogenase complex synthesized in acetate-grown cells of M. thermophila.

Acetate Kinase↗
PubMed1987

Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes.

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.

Animals↗
PubMed1987

Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme.

Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain.

Chlorella↗
PubMed1987

Anti-flavin antibodies.

Antibodies were elicited to FAD by using the hapten N-6-(6-aminohexyl)-FAD conjugated to the immunogenic carrier protein bovine serum albumin. Cross-reactivity was determined by Ouchterlony double-diffusion analysis with N-6-(6-aminohexyl)-FAD coupled to rabbit serum albumin. Anti-FAD IgG was partially purified by (NH4)2SO4 precipitation followed by DEAE-cellulose/CM-cellulose and bovine serum albumin-agarose chromatography. The partially purified anti-FAD IgG fraction failed to inhibit the catalytic activities of the flavin-containing enzymes nitrate reductase, xanthine oxidase and succinate dehydrogenase, whereas enzyme activity could be inhibited by addition of antibodies elicited against the native proteins. However, the partially purified anti-FAD IgG fraction could be used as a highly sensitive and specific probe to detect proteins containing only covalently bound flavin, such as succinate dehydrogenase, p-cresol methylhydroxylase and monoamine oxidase, by immuno-blotting techniques. Detection limits were estimated to be of the order of femtomolar concentrations of FAD with increased sensitivity for the 8 alpha-N(3)-histidyl linkage compared with 8 alpha-O-tyrosyl substitution.

Animals↗
PubMed1987

Radiation inactivation of hepatic sulfite oxidase.

Sulfite oxidase (EC 1.8.3.1), purified from chicken liver, is comprised of two identical subunits of 55 kDa, each of which contains a molybdenum and heme prosthetic group. The functional size of sulfite oxidase was determined by radiation inactivation analysis using both full, sulfite:cytochrome c reductase, and partial, sulfite:ferricyanide reductase, catalytic activities. Inactivation of full enzyme activity indicated a target size of 42 kDa while the partial activity indicated a target size of 25 kDa. These results confirm the earlier findings of two equivalent subunits and suggest the presence of a functional domain within the subunit structure that contains the molybdenum center and exhibits a smaller molecular mass than that of the enzyme subunit.

Animals↗
PubMed1987

A molybdopterin-free form of xanthine oxidase.

A previously unidentified fraction lacking xanthine:O2 activity has been isolated during affinity chromatography of bovine milk xanthine oxidase preparations on Sepharose 4B/folate gel. Unlike active, desulfo, or demolybdo forms of xanthine oxidase, this form, which typically comprises about 5% of an unfractionated enzyme solution, passes through the affinity column without binding to it, and is thus easily separated from the other species. The absorption spectrum of this fraction is very similar to that of the active form, but has a 7% lower extinction at 450 nm. Analysis of the fraction has shown that it is a dimer of normal size, but that it does not contain molybdenum or molybdopterin (MPT). The "MPT-free" xanthine oxidase contains 90-96% of the Fe found in active xanthine oxidase, and 100% of the expected sulfide. EPR and absorption difference spectroscopy indicate that the MPT-free fraction is missing approximately half of its Fe/S I centers. The presence of a new EPR signal suggests that an altered Fe/S center may account for the nearly normal Fe and sulfide content. Microwave power saturation parameters for the Fe/S II and Fe/S I centers in the MPT-free fraction are normal, with P1/2 equal to 1000 and 60 mW, respectively. The new EPR signal shows intermediate saturation behavior with a P1/2 = 200 mW. The circular dichroism spectrum of the MPT-free fraction shows distinct differences from that of active enzyme. The NADH:methylene blue activity of the MPT-free fraction is the same as that of active xanthine oxidase which exhibits xanthine:O2 activity, but NADH:cytochrome c and NADH:DCIP activities are diminished by 54 and 37%, respectively.

Animals↗