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Biomedical subjects

M T Wilson

Publications and source records attributed to M T Wilson.

At least 109 records · Page 6Linked to original sources
PubMed1990

Reactions of reduced cellobiose oxidase with oxygen. Is cellobiose oxidase primarily an oxidase?

We report rapid-mixing experiments in which cellobiose oxidase, fully reduced with cellobiose, is allowed to react with excess molecular O2. Analysis of the progress curves and their comparison with computer simulations suggests that O2 reacts only with the cytochrome b-type haem and with a rate constant of approx. 0.5 s-1. In steady state the cytochrome b is partially oxidized, whereas the flavin remains largely reduced. This situation may be contrasted with that when dichloroindophenol is substituted for O2. Under these conditions the reactions are rapid (millisecond time range), and the redox centres in the enzyme appear to be oxidized simultaneously.

Anaerobiosis↗
PubMed1989

A 1H NMR study of bovine cytochrome oxidase. Paramagnetically shifted resonances of haem a.

Dimeric and monomeric forms of mitochondrial cytochrome oxidase (EC 1.9.3.1) have been examined using 1H NMR spectroscopy. Paramagnetically shifted resonances were detected in spectra of the monomeric protein. Studies of this protein in a number of oxidation and ligation states have assigned these resonances to ferrihaem a. The temperature and pH dependence of this new probe of haem a environment is reported.

Animals↗
PubMed1988

Rapid kinetic studies of the reduction of cellobiose oxidase from the white-rot fungus Sporotrichum pulverulentum by cellobiose.

The reactions between cellobiose and cellobiose oxidase were investigated by stopped-flow spectrophotometry. Under anaerobic conditions rapid reduction of the associated flavin is followed by slower reduction of cytochrome b. The kinetic difference spectra are reported. The rate of flavin reduction depends on the cellobiose concentration (with an apparent second-order rate constant of approx. 10(5) M-1.s-1) but reaches a rate limit of approx. 20 s-1. In contrast, the rate of cytochrome b reduction decreases at high cellobiose concentrations. Kinetic titrations of the flavin and cytochrome b moieties yield the stoichiometries of the separate reactions, i.e. the number of moles of cellobiose needed to fully reduce 1 mole of each redox component. The rate constant for cytochrome b reduction, unlike that for flavin reduction, increased with enzyme concentration, prompting the conclusion that any given cytochrome b centre is reduced preferentially by flavin groups in different molecules rather than by its partner flavin within the same monomer. These data are discussed in the context of a scheme that rationalizes them and accounts for the overall stoichiometry in which three two-electron donors (cellobiose molecules) reduce two three-electron acceptors (the flavin-cytochrome b of cellobiose oxidase).

Carbohydrate Dehydrogenases↗
PubMed1988

Single cell microspectroscopy reveals that erythrocytes containing hemoglobin S retain a 'memory' of previous sickling cycles.

Red blood cells from patients homozygotes for hemoglobin S (HbS) have been studied using a computer-controlled microspectrophotometer, which allows measurements of spectra and dynamics to be undertaken in a single erythrocyte. Complete photodissociation of HbCO results in polymerization of intracellular deoxyhemoglobin S and deformation of the cell. This is associated with a delayed optical change, which, for the same cell, was found to be highly reproducible between repeated cycles of sickling. Comparison of photographic records and absorbance time courses indicates that an erythrocyte, once having undergone a photochemically induced sickling event, always deforms along the same axis during subsequent cycles. This behaviour implies that the cell retains a 'memory' of its previous cycle(s), possibly via slow relaxations of the membrane. In addition, rebinding of CO to intracellular hemoglobin was found to be slower if measured after deformation of the cell, with possible important implications for the pathological mechanism of sickling.

Anemia, Sickle Cell↗
PubMed1988

1H-n.m.r. and c.d. studies of haem orientational disorder in sperm-whale myoglobin and human haemoglobin.

1H-n.m.r. and c.d. studies on sperm-whale myoglobin show that the c.d. signal in the Soret region is inversely and linearly related to the proportion of minor isomer present. An alternative method, 'pH jump', is described for inducing orientational disorder in sperm-whale myoglobin without recourse to reconstitution. 1H-n.m.r. studies on human haemoglobin A indicate little heterogeneity in freshly isolated haemoglobin A, but the effect is enhanced in freeze-dried Sigma haemoglobin A.

Animals↗
PubMed1988

Electron transfer and conformation states in bovine cytochrome c oxidase.

The fluorophores 1,5-I-AEDANS and eosin maleimide bind to subunit III of bovine cytochrome c oxidase. Fluorescence lifetime measurements have been made of bound AEDANS under a number of conditions. It appears that the spatial relationship between this bound probe and metal centers is unaffected by the redox changes in the enzyme. Cyanide binding to CuA-modified cytochrome c oxidase during turnover suggests that reduction of cytochrome a leads to exposure of the cytochrome a3-CuB binuclear center to incoming ligands. These results are discussed in terms of a model describing the roles of cytochrome a and CuA in triggering the "closed" to "open" transition.

Animals↗
PubMed1987

Cytochrome-c oxidase. Subunit structure and proton pumping.

This article reviews the significance of the subunit structure of cytochrome-c oxidase in proton pumping and in particular summarizes available evidences for or against a role of subunit III in the control of this important function of the enzyme.

Chemical Phenomena↗
PubMed1987

Zinc cytochrome c fluorescence as a probe for conformational changes in cytochrome c oxidase.

Zinc cytochrome c forms tight 1:1 complexes with a variety of derivatives of cytochrome c oxidase. On complex-formation the fluorescence of zinc cytochrome c is diminished. Titrations of zinc cytochrome c with cytochrome c oxidase, followed through the fluorescence emission of the former, have yielded both binding constants (K approximately 7 x 10(6) M-1 for the fully oxidized and 2 x 10(7) M-1 for the fully reduced enzyme) and distance information. Comparison of steady-state measurements obtained by absorbance and fluorescence spectroscopy in the presence and in the absence of cyanide show that it is the reduction of cytochrome a and/or CuA that triggers a conformational change: this increases the zinc cytochrome c to acceptor (most probably cytochrome a itself) distance by some 0.5 nm. Ligand binding to the fully oxidized or fully reduced enzyme leaves the extent of fluorescence quenching unchanged, whereas binding of cyanide to the half-reduced enzyme (a2+CuA+CuB2+-CN(-)-a3(3+)) enhances fluorescence emission relative to that for the fully reduced enzyme, implying further relative movement of donor and acceptor.

Cytochrome c Group↗
PubMed1987

Reconstitution of monomeric cytochrome c oxidase into phospholipid vesicles yields functionally interacting cytochrome aa3 units.

When the carbon monoxide complex of fully reduced cytochrome c oxidase, reconstituted into liposomes, is mixed with oxygen-containing buffer, complex kinetic progress curves are observed. This pattern is seen irrespective of whether the oxidase used in reconstitution is the dimeric or monomeric (subunit III-depleted) enzyme. These findings are interpreted in the light of similar experiments on the detergent-solubilized enzyme reported by Gibson and Greenwood (Gibson, Q.H., and Greenwood, C. (1963) Biochem. J. 86, 541-554) and confirmed by ourselves. We conclude that reconstitution of monomeric (subunit III-less) enzyme yields, preferentially, vesicles containing more than one functional unit, possibly associated as dimers. This result is of significance to our understanding of the relationships between aggregation state and proton pumping capacity of cytochrome oxidase.

Animals↗
PubMed1987

Functional consequences of haem orientational disorder in sperm-whale and yellow-fin-tuna myoglobins.

Ligand-binding kinetics of native and reconstituted sperm-whale myoglobin were studied in relation to haem orientational disorder by rapid kinetic methods. In addition, native yellow-fin-tuna myoglobin with significant amount of haem disorder was also used. The O2 dissociation and association rates were found for the proteins with different degrees of haem disorder, and these results suggest that the isomers are characterized by almost identical kinetic parameters. Rates of CO recombination after photolysis were also identical for the two orientational isomers. The results clearly indicate that the rotation of the haem about the alpha-gamma meso axis has little or no effect on the ligand-binding properties of these myoglobins.

Animals↗
PubMed1987

Levels of glycosylated haemoglobin in the Arabian camel (Camelus dromedarius).

Proportions of glycosylated haemoglobin (Hb) were determined in 10 Arabian camels (Camelus dromedarius) and were compared with normal controls (n = 59) and diabetic patients (n = 47) using the thiobarbituric acid (TBA) method. The level of glycosylated haemoglobin (5.5%) in camels is significantly different from that of normal healthy humans (4.9%) (P less than 0.001). Whereas the glucose levels were comparable, this difference in percentages of glycosylated haemoglobin may be explained by the difference in survival time between human and camel red blood cells.

Animals↗
PubMed1987

Glycosylated hemoglobin in Saudi sickle cell patients with glucose-6-phosphate dehydrogenase deficiency.

Glycosylated hemoglobin (Hb) was measured in 42 Saudi sickle cell patients and in 38 sickle cell patients with glucose-6-phosphate dehydrogenase (G-6-PD) deficiency. A statistically significant decrease in the percentages of glycosylated Hb was found in patients with sickle cell anemia when compared with those SS subjects with G-6-PD deficiency. Since glycosylated Hb is considered as an index of the red blood cell's life span, it is suggested that the enzyme defect in these patients is possibly ameliorating the severity of sickle cell anemia.

Anemia, Sickle Cell↗
PubMed1987

Zinc and copper status in patients with sickle cell anemia.

Plasma zinc and copper concentrations were determined by atomic absorption spectroscopy in 57 patients with sickle cell anemia and in 45 control subjects from the Eastern Province of Saudi Arabia. Plasma zinc and copper levels in patients were found to be close to those of the control subjects. Similarly, there was a difference neither in urinary zinc level nor in the ratio Cu:Zn in patients and control subjects. This is in contrast to the situation which exists in North American Black subjects with sickle cell anemia, who are known to have zinc deficiency as well as a further decrease in zinc level during sickle cell crises. The near-normal levels of zinc and copper found in Saudi sickle cell patients therefore exclude zinc deficiency and confirm that this population exhibits a milder form of sickle cell anemia.

Adolescent↗
PubMed1987

Determination of glycosylated haemoglobin in normal, newborn and diabetic Saudi Arabs.

Colorimetric determinations of glycosylated haemoglobin by the thiobarbituric acid method were carried out in normal adults (n = 142), newly born infants of healthy mothers (n = 81), and in a group of patients with diabetes mellitus (n = 124). The glycosylated haemoglobin level in normal adult males was 4.9%, which is close to that reported for other populations. No correlation was found between age and the levels of glycosylated haemoglobin in males over 10 years old. However, the mean value for glycosylated haemoglobin in cord blood was 4.1%, significantly different from that of adults. The range values for glycosylated haemoglobin in diabetic patients was 7-19%. The mean value for glycosylated haemoglobin was 10.9%, similar to that established in other diabetic populations. Results of colorimetric determinations of glycosylated haemoglobin in the Saudi population compare well with other ethnic groups and, therefore, suggest no ethnic differences in glycosylated haemoglobin levels.

Adult↗
PubMed1986

Characterization of haem disorder by circular dichroism.

Native and reconstituted myoglobin were prepared and their c.d. spectra recorded in the Soret region. Time-dependent changes in dichroism following reconstitution were observed and related to haem orientational disorder. Comparative c.d. studies, in agreement with n.m.r. studies, reveal that the degree and nature of this disorder are species-dependent.

Circular Dichroism↗