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Biomedical subjects

D J Walton

Publications and source records attributed to D J Walton.

At least 19 recordsLinked to original sources
PubMed2005

Electrochemical study of silver thiosulphate reduction in the absence and presence of ultrasound.

The electrochemical reduction of silver thiosulphate was studied potentiostatically on platinum electrodes in the absence and presence of ultrasound (20 kHz). This system is irreversible and the reaction is both diffusion and kinetically controlled. The slowest step is the kinetic reaction especially the chemisorption of ions at the electrode surface. Ultrasound greatly improves the mass transport, which can be explained by changing from diffusion to mainly convection. This paper reports the effect of ultrasound upon electrode kinetic and mass-transport parameters at various RDE rotation speeds and ultrasonic intensities. It was found that the heterogeneous rate constant (kf) is improved in the presence of ultrasound due to the increase in the formal or standard heterogeneous rate constant (k0) (approximately by 10-fold under sonication).

Computer Simulation↗
PubMed2004

Reconstitution of nicotinic acetylcholine receptors into gel-protected lipid membranes.

We report the functional reconstitution of nicotinic acetylcholine receptors into gel-protected bilayer lipid membranes using two different methods. In the first case, reconstitution was achieved by direct membrane formation from an emulsion of glycerol monooleate, hexane, and a membrane receptor extract. In the second case, incorporation was achieved via the fusion of vesicles from a preparation of membrane-bound receptors into preformed membranes after diffusion through the protective front gel layer. Measurement of the dc conductivity of the membranes in the presence of either acetylcholine or alpha-bungarotoxin was used to test for the functional activity of incorporated receptors.

Electrodes↗
PubMed2004

Sonovoltammetric studies on copper in buffered alkaline solution.

The effect of ultrasound on the voltammetry of copper in alkaline solution is reported. At pH 7 the electrode surface remains active after scanning to ca. +1.0 V (vs. SCE) and the effects of ultrasound show the expected substantial enhancement in limiting current due to improved mass transport under ultrasound. However at pH 9, whereas the silent scan is only slightly altered in gross detail from that obtained at pH 7, the sonicated scan is significantly different. This shows the expected current increase only up until ca. +0.6 V (vs. SCE), where there is a substantial loss of current showing a passivation phenomenon that is enhanced by ultrasound. In addition, during the reverse (reduction) scan under ultrasound an anodic peak appears. This suggests reactivation of the electrode during the cathodic sweep, possibly by reductive removal of a transient species from the electrode/(hydr)oxide interface at a potential where oxidation still occurs. Increasing the pH to 11 further shifts the cathodic peaks in the silent voltammogram.

Journal Article↗
PubMed2002

The effect of ultrasound upon the oxidation of thiosulphate on stainless steel and platinum electrodes.

Ultrasound was found to increase the oxidation peak current and hence the decomposition rate of thiosulphate 50-fold compared to silent conditions. The effects of the ultrasonic frequency (20 and 38 kHz) and power upon the electrochemical oxidation of thiosulphate in aqueous KCl (1 mol dm-3) at stationary stainless steel and platinum electrodes were studied chronoamperometrically and potentiostatically (at various scan rates). No sigmoidal-shaped voltammograms were observed for the redox couple S4O6(2-)/S2O3(2-) in the presence of ultrasound. However, application of ultrasound to this redox couple provided an increase in the oxidation peak current at the frequencies employed, the magnitude of which varied with concentration, scan rate and ultrasonic power. Under sonication at 20 and 38 kHz, the oxidation peak potential shifted anodically with increasing ultrasonic power. This anodic shift in potential may be due to the formation of hydroxyl radicals, changes in electrode surface composition and complex adsorption phenomena. The large increase in oxidation peak currents and the rates of decomposition of thiosulphate, in the presence of ultrasound, are explained in terms of enhanced mass transfer at the electrode due to cavitation and acoustic streaming together with microstreaming coupled with adsorption phenomena. It is also shown that changes in macroscopic temperature throughout the experiment are insufficient to cause the observed enhanced diffusion.

Journal Article↗
PubMed2001

Specific electrochemical nitration of horse heart myoglobin.

Earlier findings on electronitration of hen egg-white lysozyme demonstrated a product which was mononitrated at Tyr23, by ion-exchange chromatography, absorbance at 430 nm, dithionite reduction, and Edman sequencing of a nitrated proteolytic peptide. However, the whole protein was not sequenced; therefore, although the enzyme remained active upon nitration, reaction at other residues could not be completely eliminated. This study has now been extended to the redox protein myoglobin. We demonstrate the novel electronitration (electrooxidation in the presence of nitrite) of a specific tyrosine residue in horse heart myoglobin and also in apomyoglobin. Production of the yellow chromophore, 3-nitrotyrosine (3-NT), was apparent in apomyoglobin from A430 but was masked in holomyoglobin by the Soret band. In both cases, the presence of 3-NT in the electronitrated samples was further indicated by the binding of antibody to 3-NT in Western blots. High-resolution electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry revealed a reaction product at [M + 45] (consistent with substitution of NO2 for H), indicating that the nitration reaction is the only reaction occurring which gives rise to a change in mass in the electrooxidation. Fragmentation mass spectrometry identified the nitration site as Tyr103, with no nitration at Tyr146. The procedure may be useful in preparing model nitrated proteins for the study of disease mechanisms.

Amino Acid Sequence↗
PubMed1996

The effects of tyrosine nitration on the structure and function of hen egg-white lysozyme.

We have investigated the effects of tyrosine nitration (to form the weak acid, 3-nitrotyrosine) at positions 23 or 20 plus 23, on the structure and function of hen egg-white lysozyme. Enzyme activity against Micrococcus luteus cell-wall fragments or soluble substrates exhibits two phenomena. (a) A decrease in Km and kcat for the hydrolysis of soluble oligo- and poly-saccharides, resulting in only minor changes in the catalytic efficiency (kcat/Km) upon nitration. (b) The hydrolysis of M. luteus cell-wall fragments appeared to be dominated by electrostatic interactions with the protein, giving a decrease in enzyme activity as the 3-nitrotyrosyl group became ionized. Removal of the cell-wall anionic polymer, teichuronic acid, from M. luteus abolished this effect. The 3-nitrotyrosine group was also found to act as a fluorescence quencher of exposed tryptophan residues in lysozyme.

Animals↗
PubMed1995

Catalysis of reduction of carbohydrate 2-oxoaldehydes (osones) by mammalian aldose reductase and aldehyde reductase.

In mammalian tissues, carbohydrate 2-oxoaldehydes, or 'osones', formed by cleavage of carbohydrate residues from glycated proteins, cause damage to cells and tissues by cross-linking of proteins. In the substrate specificity study reported here, we show that several osones are relatively good substrates for the reduced, unactivated form of aldose reductase (EC 1.1.1.21) from human and pig muscle, and aldehyde reductase (EC 1.1.1.2) from pig kidney, enzymes that have been well characterised both structurally and mechanistically. Since these enzymes are relatively ubiquitous, they may serve to protect a large number of tissues from damage, by catalysing the reduction of locally-produced osones. Reduction of all substrates by aldehyde reductase obeyed Michaelis-Menten kinetics. In contrast, a Hill constant of about 0.5 was obtained for aldose reductase-catalysed reduction of each of the carbohydrate 2-oxoaldehydes, and for several other substrates that were examined. Although this deviation from Michaelis-Menten kinetics has been ascribed to the presence of two forms of the enzyme, activated and unactivated, our results suggest that it is a characteristic of the unactivated form.

Aldehyde Reductase↗
PubMed1994

Electrochemical modification of lysozyme: anodic reaction of tyrosine residues.

Preparative electrooxidation of lysozyme at copper electrodes held at potentials around 1.2 V vs. a saturated calomel reference electrode induces the formation of a yellow chromophore with a concomitant decrease in the pI of the protein. Ion-exchange high-performance liquid chromatography revealed two new lysozyme species with pI values of 10.8 and 10.7 (lysozyme-11.0) which bear the chromophore. Sequence analysis of these two species showed that protein with lower pI was modified at both Tyr 23 and Tyr 20 and the other exclusively at Tyr 23. ribonuclease A, subtilisin BPN', and BSA were also found to produce the same chromophore using similar electrochemical reaction schemes. Characterization of the chromophore by a variety of techniques revealed that it is apparently 3-nitrotyrosine.

Alkalies↗
PubMed1993

Site specificity of glycation of horse liver alcohol dehydrogenase in vitro.

The site specificity of in vitro glycation of horse liver alcohol dehydrogenase (ADH) was examined and the results interpreted in terms of structural features of the enzyme molecule. In a phosphate buffer solution, glycation occurred at Lys231 (the main site of glycation in vivo), at Lys228 (which is not glycated in vivo), and at several unidentified positions. Buffer anions or NAD+ did not affect glycation of Lys231; this supported our hypothesis that the base catalyst which removes a proton from carbon 2 of a Lys231-attached aldimine is part of the ADH molecule [Shilton, B.H. & Walton, D.J. (1991) J. Biol. Chem. 266, 5587-5592]. Use of a molecular modelling programme indicated that this catalyst was likely to be the imidazole group of His348, exerting its effect through the hydroxyl of Thr347. Glycation of Lys228 occurred only in the presence of phosphate; in this case molecular modelling showed that the base catalyst could be a phosphate ion, bound to ADH at a positive region of the coenzyme binding site. NAD+ inhibited glycation of Lys228 by binding to the enzyme and restricting access to glucose.

Alcohol Dehydrogenase↗
PubMed1991

Sites of glycation of human and horse liver alcohol dehydrogenase in vivo.

Sites of in vivo glycation of human and horse liver alcohol dehydrogenase were identified by cleavage of the borotritide-treated enzyme with trypsin, followed by gas-phase sequencing of the resulting tritium-labeled glycated peptides. A blank sequencing result, i.e. failure to detect an amino acid phenylthiohydantoin after completion of an Edman degradation cycle, was ascribed to an N-(1-deoxyhexitolyl)lysyl residue, which represented a glycation site on the original enzyme subunit. In human liver alcohol dehydrogenase the sites affected were the epsilon-amino groups of lysines 10, 39, 231, 248, and 325, which were glycated to the relative extents of 10, 5, 75, 5, and 5%, respectively. The site specificity of in vivo glycation of the horse enzyme is similar; 70-75% of it had occurred at lysine 231. A computer image of the crystal structure of horse liver alcohol dehydrogenase was examined. As a result, it was proposed that the high rate of glycation at lysine 231 is due to acid-base catalysis of the Amadori rearrangement by the imidazole group of histidine 348. This hypothesis was supported by showing that imidazole groups were close to sites of glycation in several other proteins.

Alcohol Dehydrogenase↗
PubMed1989

Fructose mediated crosslinking of proteins.

10-20% of the hexose bound to human ocular lens proteins was found to be attached to epsilon-amino groups of lysyl residues via carbon 2. It was concluded that the proteins had undergone nonenzymatic reactions with endogenous fructose. This process may be important in some mammalian tissues, owing to the high crosslinking potential of fructose.

Chemical Phenomena↗
PubMed1988

Evidence for glycation of horse liver alcohol dehydrogenase in vivo.

A procedure involving HPLC of N-phenylthiocarbamyl derivatives of N-(1-deoxyhexitolyl) amino acids was used to show that borohydride-treated alcohol dehydrogenase, from horse liver, contained 0.16 mol of N epsilon-(1-deoxyhexitolyl) lysine per mol of enzyme. The identity of this compound was confirmed by mass spectrometry. It was concluded that glycation of alcohol dehydrogenase had occurred in vivo, resulting in the formation of N epsilon-(1-deoxyfructosyl) lysyl residues. The presence of the latter accounted for the retention of 14% of the enzyme by an agaroseboronate gel. These findings are interesting in view of the observation [Tsai, C. S., and White, J. H. (1983) Biochem. J. 209, 309-314] that the enzyme was activated when it was glycated in vitro.

Alcohol Dehydrogenase↗
PubMed1988

Role of fructose in glycation and cross-linking of proteins.

Incubation of carbohydrate-free human serum albumin (HSA) with fructose in an aqueous buffer at pH 7.4 resulted in glycation of epsilon-amino groups of lysyl residues. A recently developed procedure, involving analysis of hexitol amino acids by high-performance liquid chromatography of phenylthiocarbamyl derivatives, was used to show that 85% of the bound hexose was attached to protein via carbon 2 (C-2). The remainder was attached to protein via carbon 1 (C-1). When incubations were conducted with glucose under identical conditions, all the hexose was attached via C-1. Examination of human ocular lens proteins showed that the majority of the covalently bound hexose was connected to epsilon-amino groups of lysyl residues via C-1; this was attributed mainly to nonenzymatic glucosylation in vivo, which has already been documented. A significant proportion (10-20%) of the bound hexose was connected via C-2. In view of the HSA-hexose incubation results (above), this indicated that the lens proteins had reacted with endogenous fructose; i.e., they had undergone nonenzymatic fructosylation in vivo. The model protein bovine pancreatic ribonuclease A reacted with fructose and glucose at similar rates under physiological conditions. However, covalent, non-disulfide cross-linking, which could be inhibited by D-penicillamine, was induced 10 times more rapidly by fructose than by glucose. It is postulated that some of the protein cross-linking that occurs in vivo is fructose-induced. The possible significance of these processes in diabetic subjects is discussed.

Cross-Linking Reagents↗
PubMed1988

Order of enzymic incorporation of O-methyl groups into the O-methyl-D-glucose-containing polysaccharide of Mycobacterium smegmatis. A tritium-labelling study.

The order of enzymic incorporation of O-methyl groups into the O-methyl-D-glucose-containing polysaccharide (MGP) of Mycobacterium smegmatis, 3MG(J)----G(I)----G(H) ----G(G)----6MG(F)----(GMG)9(E)----[G(L)----G(D)]----G(C) ----[G(K)----G(B)]----G(A)----Ga, where G is D-glucose, 3MG is 3-O-methyl-D-glucose, 6MG is 6-O-methyl-D-glucose, and Ga is D-glyceric acid, was studied by incubating cultures of M. smegmatis with L-[3H-Me]methionine for various times. MGP was then extracted from the cells, and relative radioactivities of residues D, (E + F)average and J, or of D, E average, F, and J, were determined. Tritium-labelling of these residues increased in the reducing-to-nonreducing residue direction, the steepness of the gradient becoming more shallow with increasing incubation time. The results are consistent with a biosynthetic mechanism that involves sequential addition of O-methyl groups to residues of the pre-formed D-glucan, in the reducing-to-nonreducing residue direction.

3-O-Methylglucose↗
PubMed1987

Analysis of glycated amino acids by high-performance liquid chromatography of phenylthiocarbamyl derivatives.

A method has been developed for the analysis of hexitolamino acids formed by acid-catalyzed hydrolysis of nonenzymatically glycated proteins that have been treated with sodium borohydride. The hexitolamino acids are converted into phenylthiocarbamyl (PTC) derivatives which are analyzed by reverse-phase HPLC. The PTC derivatives of N alpha-hexitolamino acids behave like lactones, migrating on the column more slowly than the corresponding PTC-amino acids. The PTC derivatives of N epsilon-glucitol- and N epsilon-mannitol-lysine are probably free acids, since they migrate faster than PTC-lysine. The method, which can be used to determine the degree of glycation of N-terminal and lysyl residues, has been applied successfully to human hemoglobin, serum albumin, and ocular lens proteins.

Amino Acids↗