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G Williamson

Publications and source records attributed to G Williamson.

At least 145 records · Page 8Linked to original sources
PubMed1991

The purification and characterization of 4-hydroxy-3-methoxycinnamic (ferulic) acid esterase from Streptomyces olivochromogenes.

A 4-hydroxy-3-methoxycinnamic acid (ferulic acid) esterase has been purified from the extracellular broth of cultures of Streptomyces olivochromogenes after growth on oat splet xylan. The purification procedure utilizes ion exchange on DEAE-BioGel A, anion exchange on Mono Q, gel filtration and hydrophobic interaction chromatography. The purified enzyme appeared as a single band on SDS-PAGE, with an apparent Mr of 29,000. Two bands, at pI7.9 and 8.5, were observed on isoelectric focusing. With methyl ferulate as substrate, the pH and temperature optima were 5.5 and 30 degrees C respectively, with a Km of 1.86 mM and Vmax of 0.3 mumols min-1 mg-1. The purfied enzyme released ferulic acid from de-starched wheat bran only in the presence of xylanase.

Bacterial Proteins↗
PubMed1990

Production and purification of a granular-starch-binding domain of glucoamylase 1 from Aspergillus niger.

A domain of glucoamylase 1 from Aspergillus niger which binds to granular starch was produced by proteolytic digestion and purified to apparent homogeneity by extraction with corn starch followed by anion-exchange chromatography and gel filtration. The peptide has a molecular weight of 25,100, contains approximately 38% carbohydrate (w/w) and corresponds to residues 471-616 at the C-terminus of glucoamylase 1. The peptide bound to granular corn starch maximally at 1.08 nmol/mg starch. It inhibited the hydrolysis of granular starch by glucoamylase 1 but had no effect on the hydrolysis of starch in solution.

Amino Acids↗
PubMed1989

Multiple forms of glutathione S-transferase from pig liver--reaction with methyl linoleate hydroperoxides.

1. Seven isoenzyme forms of glutathione S-transferase were purified from pig liver. 2. The most basic isoenzyme reduced methyl 13-hydroperoxy-cis-9,trans-11-octadecadienoate in the absence of detergent at a higher rate (0.3 mumol/min/mg protein) than predicted from substrate solubility. 3. This demonstrates that glutathione transferase possesses some surface acting character for neutral lipid hydroperoxides.

Animals↗
PubMed1988

Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The oxidation-reduction potential of p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and Müller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1987

Pneumococcal serotypes in sputum isolates during acute respiratory illness in Edinburgh.

During the years 1978-83 serotyping was carried out on all sputum isolates of pneumococci obtained from patients in the chest wards of the City Hospital, Edinburgh. In 402 patients with acute respiratory illness the peak isolation rates occurred from January to April, and the serotype distribution was similar to that seen in previous UK studies, the commonest types being 3, 6, 9, 19, 23, and 8. The overall mortality rate was 8.7%, the serotype distribution in fatal cases reflecting the distribution of the whole group. The presence of mixed infection, predominantly with Haemophilus influenzae, was associated with a lower mortality rate of 3.5%. Nearly all patients (92%) were either elderly or had a chronic underlying disease and only one death occurred in a patient under 70 years who had no pre-existing disease. Of the pneumococcal serotypes isolated from the 292 patients with chronic chest disease, 82% are included in the new 23 valent pneumococcal vaccine and the efficacy of this needs to be assessed further in high risk patients.

Aged↗
PubMed1985

Proton nuclear magnetic resonance studies of 8 alpha-N-imidazolylriboflavin in its oxidized and reduced forms.

The oxidized and hydroquinone forms of synthetic 8 alpha-N-imidazolylriboflavin have been investigated by proton nuclear magnetic resonance spectroscopy at 360 MHz. Proton resonances due to the imidazole ring, isoalloxazine ring, and ribityl side chain have been assigned on the basis of two-dimensional 1H-1H correlated spectra (COSY), selective decoupling, and nuclear Overhauser effect difference spectra and by comparison of computer-simulated with experimental spectra. The effect of pH on the imidazolyl resonances shows a pKa for the unsubstituted imidazole nitrogen of 6.0 +/- 0.1 for the oxidized form and a value of 7.0 +/- 0.1 for the reduced form, in good agreement with the values obtained from oxidation-reduction potential data in a previous paper [Williamson, G., & Edmondson, D. E. (1985) Biochemistry 24, 7790-7797]. Slow exchange of the flavin 8 alpha-methylene and imidazolyl C(2) protons was observed at pH 6.1 but not at pH values below 4.0 for the oxidized form of the flavin. The reduced form, but not the oxidized form, of the flavin exhibits geminal coupling of the 8 alpha-methylene protons and of the C(1') methylene protons of the ribityl side chain. The magnetic nonequivalence of the protons of these two methylene groups is suggested to result from intermolecular association of the reduced flavin in aqueous solutions at the concentrations required for the spectral experiments.

Computers↗
PubMed1985

Effect of pH on oxidation-reduction potentials of 8 alpha-N-imidazole-substituted flavins.

The pKa values for the various ionic forms of 8 alpha-N-imidazolylriboflavin were determined in its oxidized and hydroquinone forms and estimated for its semiquinone form. The pH dependence of the absorption and fluorescence spectral properties and potentiometric titration data show the pKa values for the oxidized form to be 6.02 +/- 0.03 for the 8 alpha-imidazole nitrogen and 9.67 +/- 0.05 for the N(3) position of the flavin ring. The pH dependence of the oxidation-reduction potential was determined by spectrocoulometric titrations, and the data points were compared with computer-simulated plots. Two pKa values for the hydroquinone form of the flavin were determined and assigned. The pKa for the imidazole ring is found to be 6.9 +/- 0.1 and for the N(1) position of the flavin hydroquinone is found to be 5.5 +/- 0.1. Analysis of the pH dependence of the one-electron couples E2 (flavoquinone/flavin semiquinone, Flox/Fl.) and E1 (flavin semiquinone/flavin hydroquinone, Fl./Flred) resulted in an estimated pKa of 6.5 for the 8 alpha-imidazole ring in the flavin semiquinone form. These data show the possible involvement of the ionization of the 8 alpha-imidazole substituent in the redox chemistry of flavoenzymes containing either an 8 alpha-N1- or an 8 alpha-N3-histidyl-linked covalent flavin coenzyme. Future work on oxidation-reduction potentials of this class of enzymes must take into consideration the influence of the 8 alpha-histidyl substituent.

Chemical Phenomena↗
PubMed1984

Butyryl-CoA dehydrogenase from Megasphaera elsdenii. Specificity of the catalytic reaction.

The absorption coefficient of butyryl-CoA dehydrogenase from Megasphaera elsdenii at 450 nm is determined as 14.4 mM-1 X cm-1 in the CoA-free form and 14.2 mM-1 X cm-1 in the CoA-liganded form (both yellow). The latter value is considerably higher than the earlier published estimate. Phenazine ethosulphate offers great advantages over phenazine methosulphate as a coupling dye in the catalytic assay despite giving lower Vmax. values (506 min-1 as compared with 1250 min-1 under the conditions used). The phenazine ethosulphate assay is used to establish a pH optimum of 8.05 for oxidation of 100 microM-butyryl-CoA. The rates of oxidation of a range of straight-chain, branched-chain and alicyclic acyl thioesters are used to provide the following information. Only straight-chain acyl groups containing 4-6 carbon atoms are easily accommodated by the postulated hydrophobic pocket of the enzyme. C-3-substituted acyl-CoA thioesters are not oxidized at a significant rate, suggesting that the C-3 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Acyl-CoA thioesters with substitutions at C-2 are oxidized, though at a lower rate than their straight-chain counterparts. This implies that the C-2 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Saturated alicyclic carboxylic acyl-CoA thioesters with 4-7 carbon atoms in the ring are oxidized, with maximal activity for the cyclohexane derivative. This implies that optimal oxidation requires a true trans orientation of the two departing hydrogen atoms. The strain imposed by bound unsaturated alicyclic acyl thioesters strikingly perturbs the flavin visible-absorption spectrum, with the exception of the cyclohex-2-ene derivative, which forms a complex with similar spectral properties to those of the crotonyl-CoA complex. In the thiol moiety of thioester substrates the amide bond of N-acetylcysteamine is essential for both binding and catalysis. The adenosine structure contributes substantially to strong binding, but is less important in determining the catalytic rate.

Acyl Coenzyme A↗
PubMed1983

The effect on butyryl-CoA dehydrogenase of reagents specific for nucleophilic sulphur.

Removal of the green colour of butyryl-CoA dehydrogenase by reagents specific for nucleophilic sulphur is shown to involve chemical modification of the tightly bound CoA persulphide. 5,5'-Dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) de-greens the enzyme essentially irreversibly, with a stoicheiometry of approx. 1 mol/mol of FAD. A compound separated by subsequent gel filtration is eluted at the same position as a CoA-thionitrobenzoate standard. The 35S-labelled distal sulphur atom of CoA persulphide is separated from this material. The enzyme remains fully active. Phenylmercuric acetate also de-greens the enzyme. The extent to which thiols restore the green colour declines with time. Gel filtration of mercurial-treated enzyme separates low-Mr material containing a CoA moiety, an extra S atom and a phenylmercury moiety. This material, added to yellow butyryl-CoA dehydrogenase with an excess of thiol, re-forms green enzyme, but it loses this ability on storage. The results are explicable if it is assumed that the thionitrobenzoate derivative of CoA persulphide loses the extra sulphur atom much more readily than does the phenylmercury derivative.

Butyryl-CoA Dehydrogenase↗
PubMed1982

A convenient and rapid method for the complete removal of CoA from butyryl-CoA dehydrogenase.

The commercially available gel, 2-pyridyl disulphide hydroxypropyl ether-Sepharose (thiopropyl-Sepharose 6B), can be used to remove bound ligand completely from butyryl-CoA dehydrogenase (EC 1.399.2) in two simple operations. The resultant enzyme forms normal complexes with acetoacetyl-CoA and CoA persulphide, contains no bound CoA as determined by the enzymatic assay for CoA, and retains full catalytic activity.

Animals↗
PubMed1982

Evidence that the greening ligand in native butyryl-CoA dehydrogenase is a CoA persulfide.

Yellow butyryl-CoA dehydrogenase and general acyl-CoA dehydrogenase are "greened" by a mixture of coenzyme A plus elemental sulfur. The resultant stable complex contains an identical ligand with that present in native green butyryl-CoA dehydrogenase and has the same broad absorption band centered at 710 nm. Evidence is presented that the greening ligand is a CoA persulfide, possibly a mimic of the substrate carbanion thought to be generated early in the normal catalytic cycle. Variation in the position of the long wavelength band on replacement of FAD by a series of analogs of differing oxidation-reduction potential is consistent with a charge-transfer complex between a persulfide as the donor and oxidized flavin as the acceptor. The possible physiological and metabolic significance is discussed.

Acyl-CoA Dehydrogenases↗