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F M Dickinson

Publications and source records attributed to F M Dickinson.

At least 19 recordsLinked to original sources
PubMed1992

Long-chain acyl-CoA ester intermediates of beta-oxidation of mono- and di-carboxylic fatty acids by extracts of Corynebacterium sp. strain 7E1C.

beta-Oxidation of palmitate and tetradecanedioic acid was studied in cell-free extracts of the Gram-positive bacterium Corynebacterium sp. strain 7E1C, and the acyl-CoA ester intermediates formed were analysed by h.p.l.c. beta-Oxidation assays displayed a lag phase before a constant rate of NAD+ reduction was obtained. The length of the lag phase was inversely proportional to the number of units of activity added to assays. This is a characteristic feature of a system of consecutive reactions proceeding via free intermediates. During beta-oxidation of palmitate all the saturated acyl-CoAs from C16 to C8 were detected together with trace amounts of unsaturated and 3-hydroxy-intermediates. The time-course of intermediate formation again indicated a precursor-product relationship indicative of free intermediates being formed. When 3-hydroxyacyl-CoA dehydrogenase was inhibited by completely removing NAD+ from assays, the major acyl-CoAs, detected during palmitate beta-oxidation were palmitoyl-CoA, hexadeca-2-enoyl-CoA and 3-hydroxypalmitoyl-CoA. These compounds also displayed a precursor-product relationship. Under normal assay conditions the acyl-CoA dehydrogenase(s) are the probable rate-limiting enzyme(s) of the beta-oxidation spiral. These results indicate that in cell-free extracts of Corynebacterium sp. strain 7E1C, beta-oxidation proceeds via free acyl-CoA intermediates and is at variance with the concept of substrate channelling or of a 'leaky hose pipe' model as proposed for mitochondrial beta-oxidation in eukaryotic cells. The significant accumulation of chain-shortened acyl-CoA esters is similar to the situation observed for mammalian peroxisomal beta-oxidation.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1992

Purification and some properties of alcohol oxidase from alkane-grown Candida tropicalis.

Alcohol oxidase was purified to homogeneity from membrane fractions obtained from alkane-grown Candida tropicalis. The enzyme appears to be a dimer of equal-sized subunits of Mr 70000. The purified enzyme is photosensitive and contains flavin-type material which is released by a combination of boiling and proteolytic digestion. The identity of the flavin material is not yet known, but it is not FMN, FAD or riboflavin. The enzyme is most active with dodecan-I-ol, but other long-chain alcohols are also attacked. The enzyme shows a weak, but significant activity towards long-chain aldehydes. Detailed kinetic studies with decan-1-ol as substrate suggest a group-transfer (Ping-Pong)-type mechanism of catalysis.

Alcohol Oxidoreductases↗
PubMed1992

Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N.

Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD(+)-dependent enzyme and two NADP(+)-dependent enzymes. One of the NADP(+)-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent Km value of 5.2 microM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD(+)-dependent enzyme, an NADP(+)-dependent enzyme and one which was nucleotide independent. The NAD(+)-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP(+)-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent Km value of 0.2 microM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Würster's Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent Km of 0.36 mM for decanal.

Acinetobacter calcoaceticus↗
PubMed1989

The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step.

When the production of bilirubin by biliverdin reductase was monitored at 460 nm by stopped-flow spectrophotometry a 'burst' was observed with a first-order rate constant at pH 8 of 20 s-1. The steady-state rate was established on completion of the 'burst'. When the reaction was monitored at 401 nm there was no observed steady-state rate, but a diminished pre-steady-state 'burst' reaction was still seen with a rate constant of 22 s-1. We argue that the rate-limiting reaction is the dissociation of bilirubin from an enzyme.NADP+.bilirubin complex. With NADPH as the cofactor the hydride-transfer step was shown to exhibit pH-dependence associated with an ionizing group with a pK of 7.2. The kinetics of NADPH binding to the enzyme at pH 7.0 were measured by monitoring the quenching of protein fluorescence on binding the coenzyme.

Animals↗
PubMed1989

Some comparisons of pig and sheep liver cytosolic aldehyde dehydrogenases.

1. The pig enzyme was purified to homogeneity and was found to be a tetramer of apparently identical subunits. 2. The pig enzyme was found to contain 1 mol NADH/mol enzyme which is tightly bound, which is not directly involved in catalysis and which so far has not been removed from the enzyme so as to produce an active apoenzyme. 3. The pig enzyme seems to contain only one functioning active site/tetramer. 4. The pig and sheep enzymes are compared in respect of NADH binding, substrate specificity, immunological response and surface charge.

Aldehyde Dehydrogenase↗
PubMed1988

Studies on the unusual behaviour of bovine liver UDP-glucose dehydrogenase in assays at acid and neutral pH and on the presence of tightly bound nucleotide material in purified preparations of this enzyme.

Assays of UDP-glucose dehydrogenase at pH 6.0 show long (10-15 min) lag periods before the steady-state rate is established, but at pH 9.0 no lag is observed. At intermediate pH values the lag is progressively shorter as the pH becomes more alkaline. The behaviour of the enzyme in assays at neutral and acid pH depends on the pH and concentration of the enzyme used to initiate the assay. The steady-state rate at pH 6.0 is strongly concentration-dependent. It is suggested that these phenomena arise because of the slow dissociation of an inactive enzyme species to an active one. Purified preparations of the enzyme release approx. 1 mol of a UDP-sugar/mol of enzyme subunit on denaturation. The identity of the UDP-sugar is unknown.

Animals↗
PubMed1987

The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae.

The effect of K+ on assays of the enzyme was studied and it appears that the activation occurs slowly by a two-step process. Kinetic measurements suggest that the enzyme-catalysed reaction can proceed slowly (0.4%) in the complete absence of K+. The enzyme exhibits a K+-activated esterase activity, which is further activated by NAD+ or NADH. Stopped-flow studies indicated that the principal effect of K+ on the dehydrogenase reaction is to accelerate a step (possibly acyl-enzyme hydrolysis) associated with a fluorescence and small absorbance transient that occurs after hydride transfer and before NADH dissociation from the terminal E-NADH complex. The variation of activity of the enzyme with pH was studied. An enzyme group with pKa approx. 7.1 apparently promotes enzyme activity when in its alkaline form.

Acetaldehyde↗
PubMed1986

Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase. The effect of pH on the aldehyde binding reactions and a re-examination of the problem of the site of proton release in the mechanism.

Initial-rate measurements and stopped-flow spectrophotometric experiments over a wide range of pH implicate an enzyme group of pKa approximately 6.6 affecting the aldehyde binding reactions. It is possible, though not proved, that the group involved is the cysteine residue involved in catalysis. Stopped-flow fluorescence studies show that a group of pKa greater than 8.5 facilitates hydrolysis of the NADH-containing acyl-enzyme species. The identity of this group is quite unknown. Studies with 4-nitrobenzaldehyde show that this substrate gives marked substrate inhibition at quite low (less than 20 microM) concentrations. The mechanism of catalysis seems to be the same as for propionaldehyde oxidation. It is argued that proton release occurs with both substrates on hydrolysis of the NADH-containing acyl-enzyme and not before hydride transfer, as has been previously suggested [Bennett, Buckley & Blackwell (1982) Biochemistry 21, 4407-4413].

Aldehyde Dehydrogenase↗
PubMed1986

The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme.

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.

Aldehyde Dehydrogenase↗
PubMed1985

Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase.

The dissociation of the aldehyde dehydrogenase X NADH complex was studied by displacement with NAD+. The association reaction of enzyme and NADH was also studied. These processes are biphasic, as shown by McGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462], but the details of the dissociation reaction are significantly different from those given by those authors. Spectral and kinetic experiments provide evidence for the formation of abortive complexes of the type enzyme X NADH X aldehyde. Kinetic studies at different wavelengths with transcinnamaldehyde as substrate provide evidence for the formation of an enzyme X NADH X cinnamoyl complex. Hydrolysis of the thioester relieves a severe quenching effect on the fluorescence of enzyme-bound NADH.

Acrolein↗
PubMed1985

Concentration effects in assays of sheep liver mitochondrial aldehyde dehydrogenase and their interpretation in terms of enzyme dissociation.

Enzyme assays exhibit lag-phases during which the rate of acetaldehyde oxidation or p-nitrophenylacetate hydrolysis accelerates. A steady-state rate is achieved, but the time taken for this to occur increases as the enzyme concentration is decreased. With 0.009 microM enzyme this takes about 15 minutes. Analysis of experimental results suggests that the activation occurs because the tetrameric enzyme dissociates to a more active species. Gel filtration behaviour of the enzyme at different concentrations and stability tests with urea provide some general support for this hypothesis.

Aldehyde Dehydrogenase↗
PubMed1984

The removal of cytosolic-type aldehyde dehydrogenase from preparations of sheep liver mitochondrial aldehyde dehydrogenase and the unusual properties of the purified mitochondrial enzyme in assays.

The pI approximately 5.2 isoenzymes of mitochondrial aldehyde dehydrogenase were separated from the other isoenzymes by pH-gradient chromatography on DEAE-Sephacel. The pI approximately 5.2 material is immunologically identical with cytosolic aldehyde dehydrogenase. It also shows sensitivity to 20 microM-disulfiram and insensitivity to 4M-urea in assays. These and other criteria seem to establish that the material is identical with the cytosolic enzyme. Mitochondrial enzyme that had been purified to remove pI approximately 5.2 isoenzymes shows concentration-dependent lag phases in assays. These effects are possibly due to the slow establishment of equilibrium between tetramer and either dimers or monomers, with the dissociated species being intrinsically more active than the tetramer.

Aldehyde Dehydrogenase↗
PubMed1983

The coenzyme-binding characteristics of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase.

The binding of NADH and NAD+ by cytoplasmic aldehyde dehydrogenase was studied by various direct and indirect methods. At pH 7.0 at 25 degrees C there appears to be approx. 1 binding site for both nucleotides per 200 000 daltons of protein, although the NAD+-binding results are rather uncertain. Estimates of the dissociation constants of the E . NADH and E . NAD+ complexes under the stated conditions are also presented. Preparations of enzyme are sometimes found to contain significant amounts of very tightly bound NAD+ and NADH. The implications of these findings are discussed.

Aldehyde Dehydrogenase↗
PubMed1982

Effects of Mg2+, Ca2+ and Mn2+ on sheep liver cytoplasmic aldehyde dehydrogenase.

Sheep liver cytoplasmic aldehyde dehydrogenase is strongly inhibited by Mg2+, Ca2+ and Mn2+. The inhibition is only partial, however, with 8-15% of activity remaining at high concentrations of these agents. In 50 mM-Tris/Hcl, pH 7.5, the concentrations giving half-maximal effect were: Mg2+, 6.5 micrometers; Ca2+, 15.2 micrometers; Mn2+, 1.5 micrometer. The esterase activity of the enzyme is not affected by such low metal ion concentrations, but appears to be activated by high concentrations. Fluorescence-titration and stopped-flow experiments provide evidence for interaction of Mg2+ with NADH complexes of the enzyme. As no evidence for the presence of increased concentrations of functioning active centres was obtained in the presence of Mg2+, it is concluded that effects of Mg2+ (and presumably Ca2+ and Mn2+ also) are brought about by trapping increased concentrations of NADH in a Mg2+-containing complex. This complex must liberate products more slowly than any of the complexes involved in the non-inhibited mechanism.

Aldehyde Dehydrogenase↗
PubMed1982

Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase.

The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme--NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.

Aldehyde Dehydrogenase↗