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T P Singer

Publications and source records attributed to T P Singer.

At least 37 records · Page 2Linked to original sources
PubMed1993

Oxidation of tetrahydrostilbazole by monoamine oxidase A demonstrates the effect of alternate pathways in the kinetic mechanism.

The steady-state kinetics for the oxidation of 1-methyl-1,2,3,6-tetrahydrostilbazole (MTHS) by purified human liver monoamine oxidase A yielded biphasic double-reciprocal plots. Rate constants from stopped-flow studies were determined to show that the apparent stimulation at high substrate concentrations can be explained in terms of the alternate oxidative pathways available to monoamine oxidase A [Ramsay, R. R. (1991) Biochemistry 30, 4624-4629]. At low substrate concentrations, the slower reoxidation of the free enzyme (second-order rate constant was 4000 M-1 s-1) predominates, but at higher concentrations the faster reoxidation of the reduced enzyme-substrate complex (38,300 M-1 s-1) becomes significant. Computer simulation using this model predicts that similar biphasic curves could be obtained for the oxidation of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, but that nonlinearity would be obvious only at concentrations above 200 Km.

Binding, Competitive↗
PubMed1993

Is complex II involved in the inhibition of mitochondrial respiration by N-methyl-4-phenylpyridinium cation (MMP+) and N-methyl-beta-carbolines?

It has been reported that N-methyl-beta-carbolinium analogues of the neurotoxic N-methyl-4-phenylpyridinium cation (MPP+) inhibit NADH-linked mitochondrial oxidations, as well as mitochondrial respiration on succinate nearly to the same extent [Fields, Albores, Neafsey and Collins (1992) Arch. Biochem. Biophys. 294, 539-544]. Those authors further claimed that MPP+ itself also blocks respiration through succinate dehydrogenase, in addition to its well-known effect on NADH dehydrogenase (Complex I), and concluded that both effects may contribute to the development of Parkinsonian symptoms. Since N-methyl-beta-carboliniums are thought to be endogenous metabolites, these findings, if verified, would have important implications on the etiology of idiopathic Parkinsonism. We have re-examined these observations, using mitochondria after full activation of succinate dehydrogenase, as well as submitochondrial particles, in which complexities due to membrane transport are not present. We report the following observations. (1) N-Methyl-beta-carboliniums inhibit mitochondrial respiration on NAD(+)-linked substrates in a time-dependent manner, and the inhibition is potentiated by the presence of tetraphenylboron anion (TPB-), as expected for positively charged compounds. (2) Unlike MPP+ itself, however, these compounds are uncouplers at higher concentrations, so that the effects seen in State 3 cannot be assigned exclusively to inhibition of NADH oxidation. (3) The effects on succinate oxidation in mitochondria, in which the full activity of the enzyme is expressed, are 1-1.5 orders of magnitude lower than on respiration via Complex I and are thus unlikely to contribute significantly to the neurotoxicity. (4) The effect of MPP+ on mitochondrial respiration via succinate dehydrogenase is trivial, in accord with previous reports from several laboratories, but contradicting the findings of Fields et al. (cited above). (5) In submitochondrial particles the inhibition of NADH oxidation (via the complete respiratory chain) has been confirmed, but it differs markedly from the action of MPP+ in two respects. First, the enhancement by TPB- is very small; secondly, the inhibition of NADH oxidation measured using ubiquinone (Q) analogues is far lower, suggesting that Complex I is not the only target. (6) In submitochondrial particles the inhibition of succinate oxidation by either O2 or Q analogues is incomplete, trivial or absent. (7) We thus conclude that we find no basis for assigning any potential biological effect of N-methyl-beta-carboliniums to the blockade of succinate oxidation.

1-Methyl-4-phenylpyridinium↗
PubMed1993

Molecular size and flexibility as determinants of selectivity in the oxidation of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine analogs by monoamine oxidase A and B.

The introduction of a methylene bridge between the phenyl and tetrahydropyridyl moieties of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) results in increased selectivity for monoamine oxidase B (MAO B) over monoamine oxidase A (MAO A). However, lengthening of this bridge results in a total loss of selectivity. In the present study, a number of isomeric 4-naphthyl-, 4-(naphthylalkyl)-, 4-thienyl-, and 4-(thienylalkyl)tetrahydropyridines, conformationally restrained and flexible analogs of MPTP, were synthesized and evaluated as potential selective substrates of MAO A and B. In terms of the parameter (turnover number)/Km, the bulky naphthyl analogs were invariably better substrates of MAO A than kynuramine, the reference substrate for this enzyme. In addition, all naphthyl analogs, regardless of conformational mobility, were more effective substrates of MAO A than MAO B. Similarly, all thienyl analogs were found to be more effective substrates of MAO B. In contrast to the naphthalenes, the conformationally restrained thiophenes 9a and 10a were found to be poor substrates of MAO B, relative to benzylamine, the reference substrate. These results suggest that the selectivity of these compounds for either MAO A or B is determined by the complex interplay of molecular size and flexibility. In this interplay, either one of these two factors may predominate.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1993

An examination of the reliability of the radiochemical assay for monoamine oxidases A and B.

The radiochemical assay for MAO A has been compared with the polarographic and alcohol dehydrogenase-coupled assays, using 5-hydroxytryptamine (5-HT) and tyramine and the homogeneous human liver enzyme expressed in yeast, and rat liver mitochondria. Despite efforts to measure true initial rates and to avoid known sources of error in the radiochemical procedure, significantly higher rates (Vmax) and lower Km values for the substrate were obtained with the polarographic than with the radiochemical method for 5-HT and tyramine, using either highly purified enzyme or mitochondria. The rate of tyramine oxidation measured by the polarographic method and the coupled assay agreed well, however. Consequently, for kinetic and inhibition studies we recommend the polarographic method for MAO A substrates. Comparison of the radiochemical, polarographic, spectrophotometric, and coupled assays for MAO B from beef liver (mitochondria and homogeneous enzyme) showed polarography to be the method of choice on all substrates tested, although, if all known sources of error are carefully controlled, the radiochemical assay gives the same Vmax and Km values. We also report that with each substrate studied the apparent Km is much lower when mitochondria are used than when the highly purified, virtually lipid-free preparations of MAO A are studied.

Animals↗
PubMed1993

Reactivation of NADH dehydrogenase (complex I) inhibited by 1-methyl-4-(4'-alkylphenyl)pyridinium analogues: a clue to the nature of the inhibition site.

Expression of the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, following oxidation to 1-methyl-4-phenylpyridinium ion (MPP+), is believed to involve inhibition of mitochondrial electron transport from NADH dehydrogenase (complex I) to ubiquinone. MPP+ and its analogues have been shown to block electron transport at or near the same site as two powerful inhibitors of mitochondrial respiration, rotenone and piericidin A. All three types of inhibitors combine at two sites on NADH dehydrogenase, a hydrophilic and hydrophobic one, and occupancy of both sites is required for complete inhibition. Tetraphenylboron anion (TPB-) in catalytic amounts is known to increase the effectiveness of positively charged MPP+ analogues in blocking mitochondrial respiration. A part of this effect involves facilitation of the entry of MPP+ congeners into the hydrophobic site by ion pairing, as has been demonstrated in studies with submitochondrial particles (electron transport particles). This communication documents the fact that TPB-, when present in molar excess over the MPP+ analogues, reverses the inhibition. This seems to involve again strong ion pairing, removal of the inhibitory analogue from one to the two binding sites, and concentration of the inhibitor in the membrane, so that only the hydrophobic binding site remains occupied, resulting in lowering of the inhibition to 30-40%.

1-Methyl-4-phenylpyridinium↗
PubMed1992

Relation of superoxide generation and lipid peroxidation to the inhibition of NADH-Q oxidoreductase by rotenone, piericidin A, and MPP+.

The addition of NADH to submitochondrial particles inhibited by agents which interrupt electron transport from NADH-Q oxidoreductase (Complex I) to Q10 (rotenone, piericidin A, and MPP+) results in superoxide formation and lipid peroxidation. A study of the quantitative relations now shows that oxyradical formation does not appear to be the direct result of the inhibition. Although tetraphenyl boron (TPB) greatly enhances the inhibition by MPP+, it has no effect on O2. formation or lipid peroxidation. When submitochondrial particles completely inhibited by rotenone or piericidin A are treated with bovine serum albumin to remove spuriously bound inhibitor molecules without affecting those bound at the specific inhibition site, NADH-Q activity remains inhibited and lipid peroxidation occurs but superoxide formation ceases. Thus oxyradical formation may be the result of the binding of inhibitors at sites in the membrane other than those related to the inhibition of electron transport.

1-Methyl-4-phenylpyridinium↗
PubMed1992

Interaction of flexible analogs of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and of N-methyl-4-phenylpyridinium with highly purified monoamine oxidase A and B.

Sixteen analogs of N-methyl-1,2,3,6-tetrahydropyridine (MPTP) of varying degrees of flexibility have been studied as substrates of highly purified monoamine oxidases (MAO) A and B. The relative effectiveness of the various tetrahydropyridines as substrates of MAO A and B were evaluated in terms of the function turnover number/Km, as determined by initial rate measurements. The insertion of a methylene bridge between the phenyl and tetrahydropyridine moieties of MPTP to yield N-methyl-4-benzyl-1,2,3,6-tetrahydropyridine, rendering the molecule more flexible, greatly enhances reactivity with MAO B, but not with MAO A, as compared with MPTP itself, in accord with data in the literature (Youngster et al., 1989a). The ethylene-bridged MPTP analog, on the other hand, is a far better substrate of both forms of MAO than is MPTP itself. The effect of molecular flexibility on the rate of oxidation of these compounds is obscured by substituents on the aromatic ring. Branching and rigidity were detrimental to the activity as substrates of both forms of MAO. Those analogs of 1 which contain small electron-withdrawing substituents in the phenyl ring were found to be more selective for MAO B, while those substituted with bulky groups were selectively oxidized by MAO A. The substrate binding site of MAO A probably contains a lipophilic pocket larger than that found in a similar site in MAO B.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1991

Differences in substrate specificities of monoamine oxidase A from human liver and placenta.

The substrate specificities of monoamine oxidase (MAO) A isolated from human placenta and of human liver expressed in yeast have been compared in homogeneous preparations with respect to Vmax and Km values for natural and synthetic substrates and Ki values for competitive inhibitors. MAO A from these two sources is known to differ in at least 5 amino acid residues. While the Km and Ki values were found to be nearly identical in the enzymes from these two sources, the Vmax differed significantly on bulky synthetic substrates.

1-Methyl-4-phenylpyridinium↗
PubMed1991

Evidence that the inhibition sites of the neurotoxic amine 1-methyl-4-phenylpyridinium (MPP+) and of the respiratory chain inhibitor piericidin A are the same.

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), interrupts mitochondrial electron transfer at the NADH dehydrogenase-ubiquinone junction, as do the respiratory chain inhibitors rotenone, piericidin A and barbiturates. Proof that these classical respiratory chain inhibitors and MPP+ react at the same site in the complex NADH dehydrogenase molecule has been difficult to obtain because none of these compounds bind covalently to the target. The 4'-alkyl derivatives of MPP+ inhibit NADH oxidation in submitochondrial particles at much lower concentrations than does MPP+ itself, but still dissociate on washing the membrane preparations, with consequent re-activation of the enzyme. The MPP+ analogues with short alkyl chains prevent the binding of 14C-labelled piericidin A to the membrane and thus must act at the same site, but analogues with alkyl chains longer than heptyl do not prevent binding of [14C]piericidin.

1-Methyl-4-phenylpyridinium↗
PubMed1991

Interaction of 1-methyl-4-phenylpyridinium ion (MPP+) and its analogs with the rotenone/piericidin binding site of NADH dehydrogenase.

Nigrostriatal cell death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease results from the inhibition of mitochondrial respiration by 1-methyl-4-phenylpyridinium (MPP+). MPP+ blocks electron flow from NADH dehydrogenase to coenzyme Q at or near the same site as do rotenone and piericidin and protects against binding of and loss of activity due to these inhibitors. The 4'-analogs of MPP+ showed increasing affinity for the site with increasing length of alkyl chain, with the lowest Ki, for 4'-heptyl-MPP+, being 6 microM. The 4'-analogs compete with rotenone for the binding site in a concentration-dependent manner. They protect the activity of the enzyme from inhibition by piericidin in parallel to preventing its binding, indicating that the analogs and piericidin bind at the same inhibitory site(s). The optimum protection, however, was afforded by 4'-propyl-MPP+. The lesser protection by the more lipophilic MPP+ analogs with longer alkyl chains may involve a different orientation in the hydrophobic cleft, allowing rotenone and piericidin to still bind even when the pyridinium cation is in a position to interrupt electron flow from NADH to coenzyme Q.

1-Methyl-4-phenylpyridinium↗
PubMed1990

A new class of powerful inhibitors of monamine oxidase A.

It is well established that 1-methyl-4-phenylpyridinium (MPP), the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and most of its analogs are good competitive inhibitors of monoamine oxidase A, with Ki values in the micromolar range, but they inhibit monoamine oxidase B only at much higher concentrations. We report here the finding that alkyl derivatives of MPP+ substituted at the 4' position of the aromatic ring are considerably more effective reversible inhibitors of the A type enzyme, with Ki values in the nanomolar range (0.075-1.6 microM). They inhibit the B type enzyme only at 2 to 3 orders of magnitude higher concentrations (32-374 microM).

1-Methyl-4-phenylpyridinium↗
PubMed1990

Mechanism of the neurotoxicity of MPTP. An update.

This review summarizes advances in our understanding of the biochemical events which underlie the remarkable neurotoxic action of MPTP (1-methyl-4-phenyl-1-1,2,3,6-tetrahydropyridine) and the parkinsonian symptoms it causes in primates. The initial biochemical event is a two-step oxidation by monoamine oxidase B in glial cells to MPP+ (1-methyl-4-phenylpyridinium). A large number of MPTP analogs substituted in the aromatic (but not in the pyridine) ring are also oxidized by monoamine oxidase A or B, is in some cases faster than any previously recognized substrate. Alkyl substitution at the 2'-position changes MPTP, a predominantly B type substrate, to an A substrate. Following concentration in the dopamine neurons by the synaptic system, which has a high affinity for the carrier, MPP+ and its positively charged neurotoxic analogs are further concentrated by the electrical gradient of the inner membrane and then more slowly penetrate the hydrophobic reaction site on NADH dehydrogenase. Both of the latter events are accelerated by the tetraphenylboron anion, which forms ion pairs with MPP+ and its analogs. Mitochondrial damage is now widely accepted as the primary cause of the MPTP induced death of the nigrostriatal cells. The molecular target of MPP+, its neurotoxic product, is NADH dehydrogenase. Recent experiments suggest that the binding site is at or near the combining site of the classical respiratory inhibitors, rotenone and piericidin A.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1990

Evidence that the blockade of mitochondrial respiration by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) involves binding at the same site as the respiratory inhibitor, rotenone.

It has been postulated that 1-methyl-4-phenylpyridinium (MPP+) blocks mitochondrial respiration by combining at the same site as rotenone, a potent inhibitor of NADH oxidation in mitochondria, known to act at the junction of NADH dehydrogenase and coenzyme Q (CoQ). The present experiments show that MPP+ and two of its analogs indeed act in a concentration dependent manner to prevent the binding of [14C]-rotenone to submitochondrial particles (ETP) and significantly decrease the inhibition of electron transport caused by rotenone. It therefore appears that MPP+ binds at the same site as rotenone or an adjacent site, supporting the hypothesis that its neurotoxic action is due to the inhibition of mitochondrial respiration.

1-Methyl-4-phenylpyridinium↗
PubMed1990

Mechanism-based inactivation of monoamine oxidases A and B by tetrahydropyridines and dihydropyridines.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its primary oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), are mechanism-based inhibitors of monoamine oxidases A and B. The pseudo-first-order rate constants for inactivation were determined for various analogues of MPTP and MPDP+ and the concentrations in all redox states were measured throughout the reaction. Disproportionation was observed for all the dihydropyridiniums, but non-enzymic oxidation was insignificant. The dihydropyridiniums were poor substrates for monoamine oxidase A and, consequently, inactivated the enzyme only slowly, despite partition coefficients lower than those for the tetrahydropyridines. For monoamine oxidase B, the dihydropyridiniums were more effective inactivators than the tetrahydropyridines. Substitutions in the aromatic ring had no major effect on the inactivation of monoamine oxidase B, but the 2'-ethyl- and 3'-chloro-substituted compounds were very poor mechanism-based inactivators of monoamine oxidase A. It is clear that both oxidation steps can generate the reactive species responsible for inactivation.

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