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A method has been elaborated for the large scale isolation of outer membranes of beef liver mitochondria. From these monoamine oxidase was partially purified and digested with trypsin and chymotrypsin to yield peptides containing riboflavin covalently linked to the peptide chain.A pure pentapeptide of riboflavin-5'-phosphate has been obtained by a series of chromatographic procedures. Edman degradation, followed by dansylation, revealed the amino acid sequence : Ser-Gly-Gly-X-Tyr, X being the amino acid to which the 0avin is attached via the Scx-carbon of the riboflavin. This site of attachment is concluded on the basis of analysis of the optical spectra in the neutral and cationic forms of the flavoquinone and of the electron spin resonance spectrum of the free radical cation. It has been known for many years that in mitochondria from certain tissues (liver, kidney) a t least one other form of covalently bound flavin occurs which is associated with monoamine oxidase [S-111. The conclusion that the monoamine oxidase of liver and kidney contain FAD [9,12] in covalently bound form was founded on the same experimental approaches as had been used earlier in establishing the presence of covalently linked FAD in succinate dehydrogenase [13] : the flavin was not released from monoamine oxidase by any combination of denaturation methods but was released in the form of flavin peptides on proteolytic digestion [S-lo].A pure flavin peptide had not been isolated, however, nor had the'sites of attachment of the peptide on the flavin or the identity of the amino acid which provides a linkage to the flavin been established.As regards the properties of the flavin from monoamine oxidase little information is available except €or optical and fluorescence excitation spectra on which published reports are in variance. This may be in part due to the fact that existing procedures for the isolation of monoamine oxidase are such that they would be expected to extract a certain amount of succinate dehydrogenase as well and would inactivate but not necessarily remove this contaminating enzyme. Thus varying degrees of contamination with covalently linked flavin originating from succinate dehydrogenase might be present in proteolytic digests and assay of the monoamine oxidase preparation for succinate dehydrogenase activity prior to digestion would not be a reliable criterion for the absence of the enzyme. The presence of succinaie dehydrogenase contaminant in monoamine oxidase preparations could also not be ruled out on the basis of studies of the homogeneity of the sample, since monoamine oxidase is isolated in several polymeric forms [lo-12,141, ruling out the use of physical criteria of homogeneity.It seemed appropriate, therefore, to work out a procedure for the isolation of the monoamine oxidase which would yield preparations virtually free from succinate dehydrogenase. Advantage was taken of the fact that while monoamine oxidase is an outer membrane enzyme, succinate dehydrogenase is located in the inner membrane. As reported in a p...

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1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a thermal breakdown product of a meperidine-like narcotic used by drug abusers as a heroin substitute, produces Parkinsonian symptoms in humans and primates. The nigrostriatal toxicity is not due to MPTP itself but to one or more oxidation products resulting from the action of monoamine oxidase (MAO) on this tertiary allylamine. Both MAO A and B catalyse the oxidation of MPTP to the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP+), which undergoes further oxidation to the fully aromatic 1-methyl-4-phenylpyridinium species (MPP+). These bio-oxidations are blocked by selective inhibitors of MAO A and B. Additionally, MPTP, MPDP+ and MPP+ are competitive inhibitors of MAO A and B. The A form of the enzyme is particularly sensitive to this type of reversible inhibition. Both MAO A and B also are irreversibly inactivated by MPTP and MPDP+, but not by MPP+. This inactivation obeys the characteristics of a mechanism-based or 'suicide' process. The inactivation, which is accompanied by the incorporation of radioactivity from methyl-labelled MPTP, is likely to result from covalent modification of the enzyme.

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3-Dimethylamino-1-propyne irreversibly inactivates mitochondrial monoamine oxidase from bovine liver. The inactivation results in the loss of absorption in the 450-500-nm region of the flavine spectrum and a concomitant increase in absorbance at 410 nm. For the enzyme-bound adduct epsilon410 = 28000. The spectral properties of the adduct of the liver enzyme with 3-dimethylamino-1-propyne are similar to those observed when the pig kidney enzyme is inactivated with pargyline (Chuang et al. (1974), J. Biol. Chem. 249, 2381). From a proteolytic digest of the enzyme inactivated with labeled inhibitor a flavine peptide has been isolated which contains 1 mol of inactivator/mol of flavine. The chemical and spectral properties of the adduct are those of compounds containing the structure --N--CH==CH--CH==N+ less than. It was concluded that the flavine-inhibtor adduct is a N-5 substituted dihydroflavine and its structure has been determined.

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