James I. Salach

Inhibition of mitochondrial NADH dehydrogenase by pyridine derivatives and its possible relation to experimental and idiopathic parkinsonism

4-Phenyl-N-methylpyridinium (MPP+), the oxidation product of the neurotoxic amine MPTP, is considerably more inhibitory to the oxidation of NAD+-linked substrates in intact mitochondria in State 3 than is 4-phenylpyridine. On adding uncouplers, the inhibition by MPP+ progressively diminishes, while the effect of 4-phenylpyridine remains. This is in accord with the fact that MPP+ is rapidly concentrated in the mitochondria by an energy-dependent process, while 4-phenylpyridine seems to enter passively with the concentration gradient. Collapse of the electrical gradient after addition of uncouplers thus leaves the inhibition by 4-phenylpyridine unaffected but causes efflux of MPP+ from the mitochondria and a reversal of its inhibitory action. In isolated inner membranes the inhibition of NADH oxidation via the respiratory chain by 4-phenylpyridine is much greater than by MPP+. MPTP and 4-phenyl-N-methylpyridinone also inhibit more than MPP+, whereas N-methylpyridinium has relatively little effect. The block is not at the point of entry of electrons into the flavoprotein since the NADH-ferricyanide activity is not inhibited by MPP+ at Vmax.

Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPP+) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NAD+-linked substrates by MPP+

1-methyl-4-phenylpyridine (MPP+), a major product of the oxidation of the neurotoxic amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been postulated to be the compound responsible for destruction of nigrostriatal neurons in man and primates and for inhibition of mitochondrial NADH oxidation which leads to cell death. We have confirmed that 0.5 mM MPP+ inhibits extensively the oxidation of NAD+-linked substrates in intact liver mitochondria in State 3 and after uncoupling, while succinate oxidation is unaffected. However, in inverted mitochondria, inner membrane preparations, and Complex I NADH oxidation is not significantly affected at this concentration of MPP+, nor are malate and glutamate dehydrogenases or the carriers of these substrates inhibited. We report here the discovery of an uptake system for MPP+ in mitochondria which is greatly potentiated by the presence of malate plus glutamate and inhibited by respiratory inhibitors, suggesting an energy-dependent carrier. A 40-fold concentration of MPP+ in the mitochondria occurs in ten minutes. This might account for the inhibition of malate and glutamate oxidation in intact mitochondria.

Oxidation of the neurotoxic amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by monoamine oxidases A and B and suicide inactivation of the enzymes by MPTP

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a thermal breakdown product of a meperidine-like narcotic analgetic used by drug abusers as a synthetic heroin, causes Parkinsonian symptoms in humans and degeneration of the substantia nigra in monkeys. MPTP is oxidized by brain mitochondrial preparations in a process which is blocked by deprenyl and pargyline, implying catalysis by monoamine oxidase B. The present paper demonstrates that pure MAO B isolated from beef liver oxidizes MPTP 38% as fast as benzylamine with a comparable Km value. Additionally, MAO A, isolated from human placenta, oxidizes MPTP to the same product at about 12% of the rate of kynuramine, again with a comparable Km value. The latter reaction is blocked by clorgyline. Both forms of MAO are progressively inactivated by MPTP by a process which follows first order kinetics. This progressive inactivation and the fact that the activity of MAO B is not significantly regenerated following gel exclusion chromatography suggest the formation of a covalent adduct with enzyme. Thus, MPTP appears to be a suicide inactivator of MAO.

Monoamine oxidase from beef liver mitochondria: simplified isolation procedure, properties, and determination of its cysteinyl flavin content.

Abstract A novel procedure is described for the isolation of monoamine oxidase from beef liver mitochondria. The procedure involves extraction of inert protein after simultaneous digestion with phospholipases A and C, followed by extraction of the enzyme by a low concentration of Triton X-100 and polymer partition. The specific activity equals the best value in the literature, but the yield is several times higher than in published procedures. On the basis of the flavin content the molecular weight is 146,000. Gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol yields a single band of 62,000 molecular weight. Thus, it appears that the native enzyme contains two subunits not separable on polyacrylamide gels, only one of which possesses covalently linked flavin. A procedure is also described for the determination of the cysteinyl flavin content of purified preparations of the enzyme.

Reversible inhibition and mechanism-based irreversible inactivation of monoamine oxidases by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

It has been suggested (Chiba et al., Biochem. Biophys. Res. Communs. (1984) 120, 574) that the neurotoxic effects of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which causes Parkinsonian symptoms in humans and other primates, are due to compounds resulting from the oxidation of MPTP by monoamine oxidase B in the brain. We reported recently that both monoamine oxidase A and B oxidize MPTP to MPDP+, the 2,3-dihydropyridinium form and that the reaction is accompanied by time-dependent, irreversible inactivation of the enzymes. Of the two forms of monoamine oxidase, the B enzyme oxidizes MPTP more rapidly and is also more sensitive to inactivation. We now wish to report that MPTP, as well as its oxidation products, MPDP+ and MPP+, the 4-phenylpyridinium form, are also potent reversible, competitive inhibitors of both monoamine oxidase A and B, particularly the former, and that the order of inhibition for the A enzyme is MPDP+ greater than MPP+ greater than MPTP, while for the B enzyme MPTP greater than MPDP+ greater than MPP+. We further report on the spectral changes and isotope incorporation accompanying the irreversible inactivation.

Identity of the active site flavin-peptide fragments from the human "A"-form and the bovine "B"-form of monoamine oxidase.

Abstract The monoamine oxidase present in the mitochondria of human placenta was verified to be the clorygyline sensitive or A form. This property was not abolished following extensive phospholipase treatment and Triton X-100 extraction. Proteolytic digestion of the partially purified enzyme using trypsin and chymotrypsin and isolation of a peptide containing the covalently linked flavin coenzyme permitted determination of the amino acid composition and sequence of the flavin region of the catalytic site of the enzyme. The structure of the flavin peptide was found to be identical to that of the bovine liver enzyme which is clorgyline insensitive, hence, the B form. The flavin peptide segment of mitochondrial monoamine oxidase is thus conserved between the two forms and among mammalian species.

Specificity of antisera prepared against pure bovine MAO-B

Antisera have been prepared against purified bovine MAO-B that appear to react selectively with MAO-B and not MAO-A, Rabbit and mouse antisera indirectly immune precipitated [125I]bovine MAO-B using inactivated Staphylococcus aureus cells, and binding of antibodies to bovine and rat MAO-B did not inhibit enzyme activity. Two continuous rat cell lines, hepatoma line MH1C1 and glioma line C6, were used to elucidate the specificity of the antisera. MH1C1 cells, which express both MAO-A and MAO-B, showed immune-specific staining with rabbit antiserum, and staining was blocked with pure MAO-B. Further, MAO-B activity and [3H]pargyline-labeled MAO molecules could be immune precipitated from solubilized mitochondrial preparations of MH1C1 cells; and immune fixation of mitochondrial proteins following SDS polyacrylamide gel electrophoresis (SDS-PAGE) revealed staining of the MAO-B, but not of the MAO-A, flavin-containing subunit. In contrast, no immune-specific immunocytochemical staining was observed in C6 cells, which have only MAO-A activity; no MAO-A activity or [3H]pargyline-labeled MAO could be immune precipitated from solubilized mitochondrial preparations of these cells, and no stained bands were observed for mitochondrial proteins resolved by SDS-PAGE and processed for immune fixation. Further support for the selectivity of this antiserum for MAO-B comes from immunocytochemical staining of rat tissues which express varying amounts of MAO-A and MAO-B activities. Hypothalamus and liver, with high levels of MAO-A and MAO-B activities showed a large number of immunoreactive cells, whereas spleen, heart and superior cervical ganglia, with high MAO-A and low MAO-B activities showed only a few or no stained cells. Catecholamine neurons in the substantia nigra, thought to contain MAO-A, did not show immune-specific staining. Skeletal muscle cells with low MAO-A and MAO-B activities did not stain. These studies provide additional evidence that MAO-A and MAO-B are distinct molecules, differentially expressed in different cell types.

Structure of the covalently bound flavin of monoamine oxidase

Abstract Flavin peptides derived from monoamine oxidase, free from succinate dehydrogenase flavin, were obtained by digestion of outer membranes of beef liver mitochondria with trypsin and chymotrypsin and purification by various chromatographic methods. The flavin peptides show the same hypsochromic shift of the optical absorption spectrum as flavin peptides from succinate dehydrogenase: the 370 mμ band of the neutral oxidized flavin is shifted to 340 mμ, whereas the cation shows a peak at 370 mμ. The ESR spectrum of the monoamine oxidase flavin cation radical also resembles that of succinate dehydrogenase flavin in that the total width is reduced from 49 G (in riboflavin) to at least 45 G, and the line width from 3.8 G (in riboflavin) to 2.3 G. The covalently bound flavins of monoamine oxidase and succinate dehydrogenase differ, however, in that the former shows the same fluorescence intensity between pH 3.4 and 8, while the latter is quenched with a pK of 4.5 ± 0.1. These observations indicate that the FAD of monoamine oxidase is covalently linked to the peptide chain through the 8α-CH 3 group of riboflavin but histidine is not the immediate substituent, as in succinate dehydrogenase. Hydrolysis of flavin peptides from monoamine oxidase in 6 N HCl at 95° yields a derivative chromatographically distinct from free flavins which is ninhydrin-positive and thus contains an amino acid bound to the 8α-position.

Catalytically active monoamine oxidase type A from human liver expressed in Saccharomyces cerevisiae contains covalent FAD.

Monoamine oxidase type A from human liver cDNA was expressed in Saccharomyces cerevisiae. This enzymes properties with respect to Km and Ki values for kynuramine and amphetamine, respectively, were similar to values for human placental enzyme. As expected, clorgyline inhibited the yeast enzyme at lower concentrations than deprenyl. Interestingly, the FAD cofactor was covalently attached and fluorescence properties of the enzyme bound prosthetic group indicate that it is attached to a cysteine residue, the same linkage observed in other monoamine oxidases. The yield of expressed enzyme is about 15 mg/l of culture with an A600 of 15. It is suggested that covalent flavin attachment proceeds by an autoflavination mechanism.

Iron content and spectral properties of highly purified bovine liver monoamine oxidase

Abstract Centrifugation of monoamine oxidase preparations from bovine liver mitochondria in sucrose gradients enabled the removal of most of the iron containing impurities without loss of catalytic activity. The nonheme iron content of the enzyme purified with a gradient was found to be not more than 5%, and the total iron not more than 25% of the flavin content of the enzyme. Thus, iron is not a component of monoamine oxidase. Most of the iron present was in the form of cytochrome impurities, the presence of which, in less pure preparations, obscured the observation of a red semiquinone formed on reduction of the enzyme with sodium dithionite. This semiquinone was not found on substrate reduction.