When uptake of the Parkinson's syndrome inducing neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its major brain metabolite MPP+ (1-methyl-4-phenylpyridinium ion) by human platelets were compared in platelet rich plasma, a much higher rate was observed for the metabolite. The uptake process was saturable (Km = 6.8 μM; Vmax = 0.064 nmole/min/mg protein) and could be blocked by inhibitors of serotonin uptake. The accumulation of MPP+ by the platelets was accompanied by a decrease in intracellular ATP and an inhibition of mitochondrial state 3 respiration. These findings are consistent with earlier reports of the effect of MPP+ on isolated mitochondria as a potential cytotoxic mechanism, but also demonstrate that the dopamine uptake system is not the only means by which this metabolite can be efficiently transported into cells.

The search for morphological clues to the etiology of schizophrenia has led to widespread application of computed tomography (CT) scans in the examination of patients. These investigations have resulted in numerous reports over the past several years of brain atrophy and increased ventricle-brain ratios (VBR), suggestive of neuronal tissue damage, associated with the disorder. Altered activity of cellular antioxidant systems have been implicated in the neuronal cell loss that is associated with degenerative diseases of the central nervous system (CNS), but this phenomenon has not been investigated with respect to functional disorders like schizophrenia. A search for such a relationship in schizophrenics with evidence of brain atrophy has been initiated by measuring the activity of the important antioxidant enzyme glutathione peroxidase (GPx) in blood samples from a population of chronic schizophrenics and age- and sex-matched nonschizophrenic mental patients as controls. A strong negative correlation has been found between GPx activity in both isolated platelets and erythrocytes and CT scan measures of brain atrophy and VBR in the schizophrenics, but not in the control population, which exhibited comparable CT scan abnormalities. These observations suggest a unique relationship of GPx to the mechanism of tissue damage in the schizophrenics.

Phosphatidylserine (PS) has recently been reported to be a specific inhibitor of B-type monoamine oxidase (MAO-B). The effect of added PS liposomes on platelet MAO-B activity was examined in two schizophrenic groups (paranoid and a mixture of residual/ undifferentiated) and in normal controls. PS was a potent partial-mixed- uncompetitive inhibitor of the platelet enzyme, whereas other phospholipids tested were without effect. The PS concentration required for 50% inhibition was significantly higher for the paranoid relative to the residuall undifferentiated group and controls. This cor- related with a lower mean basal MAO-B activity in this group. Hill plots, as a measure of the concentration dependence of PS sensitivity, however, revealed a similarity between the two schizophrenic groups in regard to inhibitor binding properties. Mean Hill coef- ficients for both groups were significantly different from the controls. The results were consistent with an in vivo role for PS as an allosteric regulator of platelet MAO-B.

Mitochondrial monoamine oxidase (MAO), type B, has been implicated in the etiology of schizophrenia. We have found the phospholipid, phosphatidylserine (PS) to be a highly specific inhibitor of MAO-B, which has led us to postulate that the PS-MAO interaction might offer a basis for the lower MAO levels observed in platelets from certain schizophrenic populations. In this study we compared platelet MAO activity with phospholipid composition in a group of normals and chronic paranoid schizophrenics. The phospholipids in platelets and erythrocytes were extracted and separated by high-performance liquid chromatography into major classes phosphatidylcholine (PC), phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol and PS. The paranoid subjects showed statistically significantly lower MAO activity as well as higher mean levels of PS and lower levels of PC in both platelets and erythrocytes, consistent with our hypothesis. The Ca2+-stimulated synthesis of serine-lipid in platelets was also monitored by incorporation of radioisotope into lipid extracts from 14C-labelled serine substrate, and no significant differences were found between subjects groups with respect to this parameter.

The reaction of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) with monoamine oxidase from a variety of tissues including rat and monkey brain, bovine liver, and human placenta and platelets was found to yield, as a primary product, a reactive photosensitive substance with an absorbance maximum at 345 nm which is not the cation 1-methyl-4-phenylpyridinium ion previously reported as a monoamine oxidase-MPTP metabolite in vivo and in vitro. Our results suggest that the 1-methyl-4-phenyl-pyridinium ion is probably only generated in subsequent nonenzymatic transformations of this reactive monoamine oxidase metabolite. This substance was found to specifically inactivate the B-form of monoamine oxidase by a photo-induced mechanism and to react directly with NADPH and dopamine. Properties of the metabolite and potential significance of its reactions to MPTP neurotoxicity are discussed.

Selective inactivation of the multiple forms of mitochondrial monoamine oxidase (MAO) by proteases in intact and hypotonically disrupted rat liver mitochondria has been used to examine the question of differential membrane orientations of the A and B enzymes. Proteases used as probes included trypsin, beta-chymotrypsin, and the extracellular protease of Staphylococcus aureus, chosen for their different amino acid specificities. With all three proteases, no changes in the relative rates of MAO-A and MAO-B inactivation were observed after disruption of the mitochondria. Trypsin and beta-chymotrypsin gave much faster rates of MAO-A inactivation in both intact and disrupted mitochondria. The selective effect of trypsin on MAO-A was also confirmed in human placental mitochondria, which possess only A-type activity. The effectiveness of hypotonicity in disrupting the outer membrane of the mitochondria was shown by rapid protease inactivation of an intermembrane space marker enzyme, adenylate kinase (EC 2.7.4.3). Contrary to some recent reports in the literature, these findings strongly suggest that the MAO-A and MAO-B multiple-form catalytic activities do not reside on opposite faces of the membrane.

The effect of acidic phospholipids on the A and B multiple forms of membrane-bound mitochondrial monoamine oxidase has been investigated by incubating liposomes with isolated rat liver mitochondrial outer membrane preparations at lipid:protein ratios of 0.01 to 1. A strong inhibition of monoamine oxidase B was observed with phosphatidylserine and a moderate activation of monoamine oxidase A with phosphatidylinositol, while cardiolipin had no significant effect on either form. The specificity of phosphatidylserine inhibition for monoamine oxidase B was also confirmed in mitochondrial outer membrane isolated from tissues containing exclusively the A or B form of the enzyme (human placenta and bovine liver). Levels of incorporation were comparable for all the phospholipids and tissues studied and could not account for the different effects observed. Inhibition of monoamine oxidase B was found to be similar in an intact mitochondria preparation to that observed in the isolated outer membrane. A recent report of activation of both monoamine oxidase forms in delipidated whole mitochondria by the acidic phospholipids was re-examined and found to involve release of monoamine oxidase from the mitochondria. The details of the effects of phosphatidylserine and phosphatidylinositol on membrane-bound monoamine oxidase are consistent with the concept of the multiple forms as two distinct peptides, and suggest a second possible mode of in vivo regulation of substrate specificity.

Abstract: Analogs of the monoamine oxidase (MAO) inhibitor pargyline with a nitroxide free radical moiety attached through an ether linkage to the para position on the benzene ring have been prepared and reacted with solubilized MAO preparations from rat and beef brain and pig liver. These compounds behave as normal irreversible inhibitors of catalytic activity, with some preference for B‐type enzyme. When the reaction was monitored by electron spin resonance (ESR), line broadening effects indicative of binding and with an apparent relation to substrate specificity of the preparation were observed. In addition, there was a slow decrease in intensity of the ESR spectra, which could be retarded by the addition of other MAO inhibitors or increased O2 and enhanced by flavin reduction. It appears to be related to development of the irreversible phase of MAO inhibition. Signal recovery with added O2 and studies of a model reaction with free flavin, suggest the signal loss to be a line broadening effect due to interaction with an enzyme‐generated paramagnetic species rather than to direct reduction of the nitroxide radical.

Conflicting reports on the association between platelet MAO activity and schizophrenia prompted a critical review and determinations on identical samples at one laboratory in Sweden and one in the USA. Samples originated from eight schizophrenics and 27 relatives belonging to a large pedigree, thus ensuring biological homogeneity.In the USA laboratory, a significantly lower MAO activity was found in the schizophrenics when benzylamine or β–phenylethylamine was used as substrate (but not with trypt–amine), while a similar result was obtained in the Swedish laboratory when tryptamine was used (but not with benzylamine or (β–phenylethylamine). Comparisons between materials examined in different laboratories do not seem meaningful until differences in methodologies have been clarified. At present there is neither proof nor disproof of MAO being a “genetic marker” for vulnerability to the schizophrenic disorder.

Effect of temperature on human platelet MAO activity was studied by using three different substrates at a temperature range of 12–44°C. The Arrhenius plot of ln Vmax versus 1T yielded a straight line when tryptamine was used as substrate. On the other hand, a nonlinear relationship was observed with both benzylamine and PEA as substrates. This suggests that there are multiple catalytic sites or multiple forms in human platelet MAO.

Effect of oxygen on the determination of human platelet monoamine oxidase (MAO) was studied by using three different substrates at three different temperatures. MAO activity using phenylethylamine as substrate increased about two times with oxygenation at all three temperatures. On the other hand, there was virtually no alteration in activity using benzylamine as substrate at any temperature. Using tryptamine as substrate, the rate of increase in MAO activity varied with the temperature used. Our findings suggest that the catalytic site(s) of the human platelet enzyme responds differentially to various substrates at different temperatures under different concentrations of oxygen.

Spin-labeled hydroxyamphetamine, a competitive reversible inhibitor of brain monoamine oxidase, has been shown to be useful as an electron spin resonance (ESR) probe of the microenvironment of the active sites of the possible monoamine oxidase multiple forms. The ESR spectrum of spin-labeled hydroxyamphetamine was strongly quenched upon binding to the enzyme. The conformation of the active site of rat brain monoamine oxidase existing in various physical states, i.e. monoamine oxidase in situ (intact brain mitochondria), crude solubilized monoamine oxidase (MAOS) and isolated monoamine oxidase fractions (MAOa and MAOb) were critically and systematically examined. Nonlinear least squares regression analyses have been used to fit the binding data (obtained at room temperature with varying spin-labeled hydroxyamphetamine concentrations) to three groups of independent noninteracting ligand-binding models. A Gibbs-Helmholtz relationship was applied to the interpretation of the measured apparent association constant K as a function of temperature ranging from 4-50 degrees with increments of 2 degreesmfrom the extracted intensive parameters, k (intrinsic association constant) and deltaF (intrinsic free energy), as well as the apparent heat, deltaH, it was clear that the microenvironment of the binding sites existing in the more purified enzyme fractions MAOa and MAOb were similar to those found in the crude solubilized enzyme. More importantly, they correlated well with the conformation of the sites characterized in situ. The data suggested that the microenvironment of this multienzyme system was unperturbed in spite of the treatment due to the isolation process. In terms of the composition of binding sites, MAOa appeared to be heterogeneous while MAOb appeared to be more homogeneous. Since the isolated fractions MAOa and MAOb possessed marked different substrate specificities, these observations directly implied that monoamine oxidase multiple forms do exist in situ. The extracted extensive parameters, n (specific binding activity, nanomoles/mg of protein), as well as the measured characteristic transition temperatures, indicated that the relative abundance of the sites which directly affected substrate specificities was indeed altered. The consistency of the characteristic transition temperatures of 21 degrees and 38 degrees for the case of intact membrane preparations was particularly significant. A tenable hypothesis is that the manipulation in the composition of the monoamine oxidase binding forms through intimate lipid-protein interactions, which has been amply demonstrated in many biomembrane systems to be functionally important might be the underlying regulatory mechanism in vivo.

Monoamine oxidase (MAO) in crude mitochondrial preparations from rat brain was solubilized, and different MAO‐active fractions were separated by agarose columns and by Sephadex electrophoresis. Any combination of these techniques yielded at least three fractions possessing MAO activity as measured by assays using radioactive serotonin and benzylamine as substrates. The molecular weight of one of the MAO forms was found to be approximately 400,000 daltons while another was at least 1.5 × 106 daltons. The crude mitochondria1 MAO was inhibited by [14C]‐labelled pargyline and then solubilized and the radioactivity of the soluble and particulate MAO was compared to the enzyme activity found in the soluble and particulate fractions. Our studies suggest that appreciable MAO activity is lost upon solubilization and that the conformation of MAO may be altered.