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K F Tipton

Publications and source records attributed to K F Tipton.

At least 19 recordsLinked to original sources
PubMed1992

Uptake and accumulation of 1-methyl-4-phenylpyridinium by rat liver mitochondria measured using an ion-selective electrode.

The compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes selective destruction of nigrostriatal dopaminergic neurons in primates, giving rise to a condition resembling Parkinson's disease. The toxicity of MPTP is believed to be due to its metabolite 1-methyl-4-phenylpyridinium (MPP+). MPP+ is an inhibitor of mitochondrial respiration at the NADH-ubiquinone oxidoreductase site and this, together with its selective transport into dopaminergic nerve terminals, accounts for its neurotoxicity. In this paper an electrode selective for MPP+ was developed and used to measure the rate of uptake and the steady-state accumulation of MPP+ in rat liver mitochondria. The initial rates of MPP+ uptake were not saturable, confirming previous work that the transport of MPP+ is not carrier-mediated. The membrane potential of mitochondria respiring on succinate was decreased by MPP+ and the steady-state accumulation ratio of MPP+ did not come to equilibrium with the mitochondrial transmembrane potential gradient (delta psi). The effect of the cation exchanger tetraphenylboron (5 microM) was to increase the initial rate of MPP+ uptake by about 20-fold and the steady-state accumulation by about 2-fold. This suggests that there may be a mechanism of efflux of MPP+ from mitochondria which allows MPP+ to cycle across the membrane and thus decrease delta psi. These data indicate that MPP+ interacts with mitochondria independently of its inhibition of NADH-ubiquinone oxidoreductase, and these alternative interactions may be of relevance for its mechanism of neurotoxicity.

1-Methyl-4-phenylpyridinium↗
PubMed1992

Steady-state kinetic analysis of aldehyde dehydrogenase from human erythrocytes.

The steady-state kinetics of purified cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from human erythrocytes have been studied at 37 degrees C. Previous studies of the enzyme from several mammalian sources, which used a lower assay temperature, have been difficult to interpret because of the substrate activation by acetaldehyde which led to complex kinetic behaviour. At 37 degrees C the initial-rate data do not depart significantly from Michaelis-Menten kinetics. Studies of the variation of initial rates as a function of the concentrations of both substrates and studies of the inhibition by NADH were consistent with a sequential mechanism being followed. High-substrate inhibition by acetaldehyde was competitive with respect to NAD+. The enzyme was not inhibited by the product acetate and thus the results of these studies, although consistent with an ordered mechanism in which NAD+ was the first substrate to bind, were inconclusive. That such a mechanism was followed was confirmed by determination of the initial-rate behaviour in the presence of acetaldehyde and glycolaldehyde as alternative substrates. When the reciprocal of the initial rate of NADH formation was plotted against the acetaldehyde concentration at a series of fixed ratios between that substrate and glycolaldehyde, a linear 'mixed inhibition' pattern was obtained, confirming the mechanism to be ordered with NAD+ being the leading substrate and with kinetically significant ternary complex-formation.

Acetaldehyde↗
PubMed1992

Kinetic and inhibition studies on catechol-O-methyltransferase affinity labelling by N-(3,4-dihydroxyphenyl)maleimide.

Initial velocity and product inhibition studies have been performed on soluble catechol-O-methyltransferase which has been partially purified from pig liver. The results are consistent with an ordered reaction mechanism, in which S-adenosyl-L-methionine (AdoMet) is the leading substrate. The enzyme is irreversibly inhibited by maleimide derivatives in a biphasic manner, which suggests a differential reaction with two thiol groups. N-(3,4-Dihydroxyphenyl)maleimide, which has a reactive moiety (maleimide ring) and an affinity moiety (catechol ring), acts as an affinity labelling compound on the more reactive SH group; AdoMet and Mg2+ protect against this modification. Total protection of this SH group results in a pseudo-first-order inhibition of the enzyme, with the apparent rate constant being proportional to the inhibitor concentration. All the other maleimide derivatives studied inhibited the enzyme by reacting with one of the two SH groups in a non-specific manner. The reaction of the other, more reactive, SH group was either specific (active-site-directed) or non-specific, depending on the substituent present in the affinity moiety and also on the length of an intermediate chain of methylene groups present between this moiety and the reactive maleimide ring. In the presence of both AdoMet and Mg2+, 3,5-dinitrocatechol, a reversible inhibitor of the enzyme which is competitive with respect to the catechol substrate, protects the enzyme from inactivation by any of the maleimide derivatives. The adducts of these maleimide derivatives formed with dithiothreitol inhibit the enzyme reversibly, showing inhibition patterns that are consistent with the mechanism deduced from the initial velocity and product inhibition studies.

Affinity Labels↗
PubMed1992

Effects of chronic ethanol feeding on rat liver mitochondrial energy metabolism.

Chronic alcohol consumption is known to decrease hepatic mitochondrial respiration rate. It was shown here that the proton leak through the mitochondrial inner membrane was unaffected by chronic ethanol treatment. This indicates that changes in proton leak are not responsible for the alterations in respiration found in mitochondria isolated from ethanol-treated rats. Therefore, the lowered coupled respiration rate is solely due to a decrease in the activity of the electron transport chain. However, this alteration was only evident in coupled respiration (i.e. state 4) and was not apparent in uncoupled respiration. Thus, chronic ethanol treatment decreases the activity of the mitochondrial electron transport chain components which have control over coupled, but not uncoupled, respiration. Mitochondrial energy metabolism is regulated by thyroid hormone status. It was shown that the chronic alcohol treatment did not affect the circulating levels of thyroxine. Furthermore, the activity of mitochondrial alpha-glycerophosphate dehydrogenase, which is strongly affected by thyroid hormones, was unaltered by alcohol treatment. Thus, the effects of ethanol treatment on mitochondria occur independently of changes in circulating thyroid hormone levels.

Animals↗
PubMed1992

Oxidative ring-coupling of tyrosine and its derivatives by purified rat intestinal peroxidase.

Intestinal peroxidase was shown to catalyse the oxidative ring-coupling of tyrosine, alpha-methyltyrosine, tyramine and morphine whereas amphetamine was not oxidized to any detectable extent. The oxidative ring-coupling reaction can be monitored by changes in absorbance spectra and the dimers formed in this way with morphine and alpha-methyltyrosine were identified by mass spectrometry. Intestinal peroxidase also catalysed the peroxidatic oxidation of L-DOPA and alpha-methyl-L-DOPA, but in this case the reaction would be expected to be more complicated and to yield a variety of possible products. The kinetic parameters for the oxidation of each of these substrates were determined. Since the products of the oxidative ring-coupling reactions may have different pharmacological properties to those of the parent compounds, these studies suggest that, in the presence of an adequate supply of metabolically produced hydrogen peroxide, the action of intestinal peroxidase may affect the behaviour and pharmacokinetics of these compounds after oral administration.

Amphetamine↗
PubMed1992

Interactions of the neurotoxin MPTP and its demethylated derivative (PTP) with monoamine oxidase-B.

The kinetics of the interactions of MPTP and its N-des-methyl-derivative (PTP) have been studied. Both were mechanism-based inhibitors as well as substrates for the enzyme. Analysis of the reaction progress-curves for the formation of the corresponding dihydropyridine derivatives allowed the kinetic parameters for the process and the partition ratio, which corresponds to the number of mol. of product formed per mol. of enzyme inactivated, to be determined for both compounds. The conversion of MPTP to its corresponding pyridinium-ion derivative through the action of MAO-B is known to be essential for its neurotoxicity. PTP has been reported not to be neurotoxic, although it appears to be a relatively good substrate for MAO-B as well as acting as a mechanism-based inhibitor. Studies of the changes in absorbance spectra during the MAO-B catalysed oxidation were consistent with the formation of the corresponding pyridinium-ion derivative (MPP+), which is known to be the effective neurotoxin, as the end-product when MPTP was oxidized. In contrast the oxidation of PTP appeared to stop at the dihydropyridine stage with no significant further oxidation to the corresponding pyridine-derivative.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1992

Developmental aspects of the monoamine-degrading enzyme monoamine oxidase.

In the rat heart, monoamine oxidase (MAO)-B activity was shown to predominate in 2- to 3-week-old animals, whereas MAO-A activity was reported to be very low in newborn rats and to increase considerably with age until it predominates. These results are in contrast with those found in the mouse heart, where an age-dependent increase in MAO-B activity with no changes in 5-hydroxytryptamine deaminating activity was found to occur. There is evidence that the adult values of MAO activity are reached early in development in rat kidney and liver. In the rat lung the adult values of MAO-A activity are reached by day 40, whereas MAO-B activity is still increasing by day 80. Important differences have been reported in the developmental pattern of the two forms of MAO in the rat and mouse brain, with a decrease in the MAO-A/MAO-B ratio during postnatal development. In the human brain, the ontogenetic development of MAO-A and MAO-B appears to parallel that observed in the rodent brain. It is worth noting that most of the available data have to be considered with reservation owing to many methodological problems. Further studies are clearly needed to get reliable information on the ontogenesis of MAO in mammalian tissues.

Animals↗
PubMed1991

The inhibition of glutamine synthetase in rat corpus striatum in vitro by methionine sulfoximine increases the neurotoxic effects of kainate and N-methyl-D-aspartate.

Coronal slices of rat brain were incubated in Krebs bicarbonate medium containing kainate (300 microM), or N-methyl-D-aspartate (500 microM). Degeneration of striatal neurons by both these toxins was apparent after 40 min incubation, and was accompanied by a 33% (kainate) and 21% (N-methyl-D-aspartate) reduction in striatal glutamine synthetase activity. Pre-incubation of the slices with 500 microM L-methionine sulfoximine, an inhibitor of glutamine synthetase, for 20 min prior to the exposure to either kainate or N-methyl-D-aspartate, again showed extensive degeneration of striatal neurons, and a supra-additive reduction in glutamine synthetase activity in the tissue. The activity of the neuronal marker enzyme, neuron-specific enolase, was also reduced by pre-incubation of the slices with L-methionine sulfoximine before the addition of kainate or N-methyl-D-aspartate, but to a much lesser extent than glutamine synthetase. The results are discussed in terms of a possible mechanism of interaction between either kainate or N-methyl-D-aspartate, and glial cell metabolism.

Animals↗
PubMed1991

The effects of assay temperature on the complex kinetics of acetaldehyde oxidation by aldehyde dehydrogenase from human erythrocytes.

Several studies have shown preparations of the cytosolic aldehyde dehydrogenase (EC 1.2.1.3) from sheep and human liver and from human erythrocytes to exhibit complex kinetic behaviour in which the dependence of the initial velocity on the concentration of acetaldehyde gives rise to downwardly curving double-reciprocal plots. This behaviour has often been analysed in terms of a sharp discontinuity in the double-reciprocal plots and its possible implications for the oxidation of acetaldehyde and other pharmacologically important aldehydes has been a subject of speculation. In the present work, it is shown that the purified, apparently homogeneous, enzyme from human erythrocytes exhibits such complex kinetic behaviour when initial rates are determined at 25 degrees, although the double-reciprocal plots describe a smooth curve with no sharp discontinuity. However, when the assays were performed at 37 degrees there was no significant deviation from Michaelis-Menten kinetics over a wide range of acetaldehyde concentrations (0.2-30 mM). At higher concentrations of acetaldehyde inhibition occurred which was competitive with respect to NAD+. These results, which indicate that the complex kinetic behaviour of aldehyde dehydrogenase is not important at physiological temperature, are interpreted in terms of the mechanisms that have been advanced to explain the phenomena.

Acetaldehyde↗
PubMed1991

The inhibition of monoamine oxidase by brofaromine.

The inhibition of rat liver mitochondrial monoamine oxidase-A (MAO-A) by brofaromine was time-dependent at low enzyme and inhibitor concentrations. The apparent sensitivity to inhibition decreased when the concentration of the mitochondrial preparation was increased. After preincubation of the enzyme with brofaromine repeated washing of the preparation, by sedimentation and resuspension, resulted in a gradual recovery of activity. This occurred more slowly than was the case when the reversible inhibitor amphetamine was used. After incubation with radioactively-labeled brofaromine the loss of radioactivity also occurred slowly. After incubation with radioactively-labeled pargyline polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphage (SDS-PAGE) showed the radioactivity to be associated with a peptide of approximate Mr 50,000, corresponding to the subunit of MAO. Pretreatment with unlabeled pargyline depressed this labeling by pargyline, indicating the latter compound to bind to the active-site of the enzyme. Labeling experiments with radioactive brofaromine indicated that there was a high degree of non-specific binding but that no significant radioactivity remained associated with the enzyme on SDS-PAGE. Chromatographic techniques and determination of H2O2 liberation indicated that, in liver there was no appreciable metabolism of brofaromine under the conditions used in the inhibition experiments. These data indicate brofaromine to be a tight-binding, but reversible inhibitor of MAO.

Animals↗
PubMed1991

Is the oxidation of milacemide by monoamine oxidase a major factor in its anticonvulsant actions?

The anticonvulsant drug milacemide (2-n-pentylaminoacetamide) is known to be oxidized by monoamine oxidase-B to yield glycinamide which then breaks-down to give glycine. It has been postulated that it is this liberation of glycine in the brain that accounts for the anticonvulsant effects. In order to test this hypothesis, and since amines bearing a methyl-group in the alpha-position have been shown to be resistant to oxidation by monoamine oxidase, the effects of milacemide were compared with those of alpha-methyl-milacemide. Although the latter compound was found to be toxic at higher concentrations, it was found to antagonize bicuculline-induced convulsions in mice. When milacemide was administered to mice (0.5 mmol/kg, p.o.) there was a substantial increase in urinary glycinamide excretion. No such increase was observed after the administration of the same dose of alpha-methyl-milacemide. Furthermore, alpha-methyl-milacemide was not oxidized by either monoamine oxidase-A or -B in vitro to any detectable extent, although it was a competitive inhibitor of both forms of the enzyme. The findings that alpha-methyl-milacemide has anticonvulsant properties in the bicuculline test but is not a substrate for monoamine oxidase or a source of urinary glycinamide cast doubt on the importance of the oxidation or milacemide to form glycinamide as a major factor in its anticonvulsant action.

Acetamides↗
PubMed1991

The oxidation of dopamine by the semicarbazide-sensitive amine oxidase (SSAO) from rat vas deferens.

The activities of monoamine oxidase A and B and the semicarbazide-sensitive amine oxidase from rat vas deferens were compared towards benzylamine and dopamine. The selective inhibitors (-)-deprenyl and clorgyline were used to allow the contributions of the A and B forms of monoamine oxidase to be determined separately. Comparison of the kinetic constants of the three enzymes towards dopamine indicated that, although each of them had activity towards this substrate, their relative contributions to the total oxidative deamination would depend on the substrate concentration. At all concentrations in the range 1 microM to 10 mM monoamine oxidase-B would contribute about 50% of the total activity. In the range 1 to 10 microM the contributions made by activities of monoamine oxidase-A and the semicarbazide-sensitive enzyme were similar but at higher concentrations the activity of the latter enzyme became more important, its contribution to the total activity rising to some 35% of the total at 500 microM dopamine. The activity of the semicarbazide-sensitive enzyme towards dopamine might thus be important under conditions where either or both the monoamine oxidases were inhibited in pharmacological studies. Its possible relevance to Norrie disease, in which both forms of the human enzyme are deficient, requires further examination.

Amine Oxidase (Copper-Containing)↗
PubMed1991

The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities.

Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.

Amino Acid Sequence↗