Twenty analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were tested for their capacity to be oxidized by pure monoamine oxidase-A (MAO-A) prepared from human placenta and pure monoamine oxidase-B (MAO-B) prepared from beef liver. Several of the MPTP analogs were very good substrates for MAO-A, for MAO-B, or for both and had low Km values and high turnover numbers. These values were similar to or even better than those of kynuramine and benzylamine, good substrates for MAO-A and MAO-B, respectively. MPTP had relatively low Km values for oxidation by both MAO-A and MAO-B. In contrast, the turnover number for MPTP oxidation by MAO-B was considerably higher than the value for MAO-A. The corresponding pyridinium species of MPTP and several of the MPTP analogs inhibited MAO-A competitively with Ki values at micromolar concentrations; in contrast the pyridinium species inhibited MAO-B competitively at considerably higher concentrations (i.e., 100 microM or greater Ki values). The data provide information concerning the structural requirements for the oxidation of tetrahydropyridines by MAO-A and MAO-B and the inhibition of these enzymes by pyridiniums.

Nineteen structural analogs of 1-methyl- Studies over the past 5 years in our laboratories and by others have provided substantial support for the hypothesis that the expression of the neurotoxicity of 1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine (MPTP) and the Parkinsonism it produces involves the following steps, all of which occur in the central nervous system. The initial bioactivation of M1PTP is catalyzed by monoamine oxidase (MAO), yielding 1-methyl-4-phenylpyridinium (MPP+) as the ultimate oxidation product (1, 2). In the case of 2'-substituted neurotoxic analogs of MPTP, part or all of the bioactivation is mediated by 4 The present paper concentrates on the terminal events leading to nigrostriatal cell death: the inhibition of NADH oxidation in mitochondria and in inverted inner membranes by MPP+ analogs and the energy-dependent accumulation of some of these analogs by intact mitochondria. The experiments reported may shed light not only on the mechanism of site 1 inhibition of mitochondrial respiration by the bioactivation products of MPTP but also on the nature of the inhibition site that MPP+ shares with rotenone, piericidin A, and barbiturates (10).MATERIALS AND METHODS Rat liver mitochondria were prepared from Sprague-Dawley rats by the mannitol procedure (11). Oxygen consumption was measured polarographically. Since inhibition of mitochondrial respiration by MPP+ and its analogs is a function of both the concentration of inhibitor and the time of its preincubation with the mitochondria, IC50 or Ki values cannot be calculated in a simple manner. Hence, the following procedure was adopted. The mitochondria, buffer, and glutamate and malate were preincubated in the polarographic chamber at 250C with several concentrations of the inhibitor for times varying from 1.5 to 9 min. At that time, ADP was added and the rate of02 consumption was determined at 250C as described (9). The results were then calculated from a plot of 1/time (min) of preincubation with the inhibitor required to reach 50% inhibition of state 3 respiration against the concentration of the inhibitor. The slope of the resulting line is

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