The monoamine oxidases play a vital role in the metabolism of biogenic amines in the central nervous system and in peripheral tissues. Using oligonucleotide probes derived from three sequenced peptide fragments, we have isolated cDNA clones that encode the A and B forms of monoamine oxidase and have determined the nucleotide sequences of these cDNAs. Comparison of the deduced amino acid sequences shows that the A and B forms have subunit molecular weights of 59,700 and 58,800, respectively, and have 70% sequence identity. Both sequences contain the pentapeptide Ser-Gly-Gly-Cys-Tyr, in which the obligatory cofactor FAD is covalently bound to cysteine. Based on differences in primary amino acid sequences and RNA gel blot analysis of mRNAs, the A and B forms of monoamine oxidase appear to be derived from separate genes.Monoamine oxidases A and B [MAO A and MAO B, respectively; amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4] in the central nervous system and in peripheral tissues catalyze the oxidative deamination of neuroactive and vasoactive amines (1) and the oxidation of xenobiotics, including the parkinsonism-producing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (2, 3). These enzymes, which are integral proteins of the outer mitochondrial membrane (4), are distinguished by differences in substrate preference (5), inhibitor specificity (6), tissue and cell distribution (7), and immunological properties (8,9). MAO A preferentially oxidizes the biogenic amine serotonin and is inactivated irreversibly by the acetylenic inhibitor clorgyline. MAO B preferentially oxidizes phenylethylamine and benzylamine and is inactivated by the irreversible inhibitors pargyline and deprenyl. The level of MAO activity in almost all human tissues consists of a mixture of both forms of the enzyme, but placental tissue contains predominantly MAO A (10), whereas platelets and lymphocytes express only MAO B (11,12). MAO A and B from several tissue sources and species appear to consist of two subunits with approximate molecular masses of 60 kDa (13,14 (15,19). Peptide maps obtained from proteinase digestion of [3H]pargyline-labeled crude or partially purified MAO A and B suggest that these enzymes differ in their amino acid sequences (17,20). Furthermore, differences in degrees of photo-dependent inactivation of these two enzymes suggest the existence of conformational or structural differences in their active sites (21-23).To clarify the molecular basis of structural and functional differences between these important enzymes, we have isolated and characterized cloned cDNAsI encoding these proteins. The nucleotide and deduced amino acid sequences for human liver MAO A and B show that these two proteins are derived from separate genes. MATERIALS AND METHODSConstruction and Screening of the Human Liver cDNA Library. A Agt1O library was constructed from poly(A)+ mRNA isolated from human liver (24). The phage library contained 2 x 106 individual clones of which 5 x 105 clones were subjected to hy...

No abstract

In the monkey dorsal raphe, we reported that 1-month (mo) of estrogen replacement, with or without progesterone supplementation for 14 days, significantly increased tryptophan hydroxylase-1 (TPH-1) mRNA; decreased serotonin reuptake transporter (SERT) mRNA and decreased monoamine oxidase (MAO)-A mRNA, but had no effect on MAO-B mRNA. Here, we questioned what effect would 1 or 5 mo of ovarian hormones or the selective estrogen receptor modulator (SERM), raloxifene, have on TPH protein and phosphorylation, and on protein expression of SERT, MAO-A or MAO-B? Raloxifene antagonizes estrogen in breast or uterus, but estrogen-like activities in the brain have been reported. Cytoplasmic and membrane extracts of the dorsal raphe region were processed for Western blotting. TPH, phosphoserine, SERT, MAO-A, and MAO-B were detected with specific antibodies. The optical densities of the signals were measured with NIH image and analyzed by ANOVA. Both 1 and 5 mo of estrogen, with or without progesterone, and 5 mo of raloxifene significantly increased TPH protein. Administration for 5 mo of estrogen plus progesterone and raloxifene also increased TPH phosphorylation. Estrogen, with or without progesterone, for 1 mo had no effect on SERT protein. However, 5 mo of estrogen and 5 mo of raloxifene increased SERT protein. Estrogen alone or combined with progesterone for 1 mo caused a significant reduction in MAO-A. Yet, after 5 mo of the same treatments, MAO-A was not different from spayed controls. Estrogen alone had no effect on MAO-B. However, the addition of progesterone significantly increased MAO-B. Raloxifene for 5 mo had no effect on MAO-A or MAO-B. Thus, to various extents, estrogen, progesterone, and raloxifene may increase serotonin production and transport. The expression of the degradative enzymes suggests a complex combination of gene transcription, post-transcriptional processing, and substrate feedback mechanisms.

Monoamine oxidases (MAO; EC 1.4.3.4.) A and B occur in the outer mitochondrial membrane and oxidize a number of important biogenic and xenobiotic amines. Monoclonal antibodies specific for human MAO A or B and immunocytochemical techniques were used to visualize the respective enzymes in human placenta, platelets, lymphocytes, liver, brain, and a human hepatoma cell line. MAO A was observed in the syncytiotrophoblast layer of term placenta, liver, and a subset of neurons in brain, but was not observed in platelets or lymphocytes, which are known to lack type A enzyme. MAO B was observed in platelets, lymphocytes, and liver, but not in placenta, which contains little or no MAO B. MAO B was also observed in a subset of neurons in the brain that was distinct from that which contained MAO A. MAO A and MAO B were also observed in some glia. Unlike most tissues examined, liver cells appeared to contain both forms of the enzyme. These studies show that MAO A and MAO B can be specifically visualized by immunocytochemical means in a variety of human cells and tissues and can provide a graphic demonstration of the high degree of cell specificity of expression of the two forms of the enzyme.

No abstract

A series of 1,2,3,4-tetrahydro-, 3,4-dihydro-, and fully aromatic isoquinolines were tested as substrates and/or inactivators of highly purified human monoamine oxidase A and B (MAO A and B). None were found to be a substrate for either enzyme, but many of these isoquinolines could selectively inhibit either MAO A or B. Stereoselective competitive inhibition of MAO A was found with the R enantiomer of all the stereoisomers tested, including salsolinol (Ki = 31 microM), salsoline (Ki = 77 microM), salsolidine (Ki = 6 microM), and carnegine (Ki = 2 microM). As a class, the 3,4-dihydro-isoquinolines were the most potent inhibitors tested (Ki = 2-130 microM), and the fully aromatic isoquinolines had intermediate activity (Ki = 17-130 microM) against MAO A. In contrast, only a few of these compounds markedly inhibited MAO B. 1,2,3,4-Tetrahydroisoquinoline, its 2-methyl derivative, and o-methylcorypalline gave apparent Ki values of 15, 1, and 29 microM, respectively, and two 3,4-dihydroisoquinolines (compounds 22 and 25) showed substantial inhibition of MAO B (Ki = 76 and 15 microM, respectively). These results support the concept that the topography of the inhibitor binding site differs in MAO A and B.

Monoamine oxidases A and B (MAO-A and MAO-B) oxidatively deaminate neurotransmitter and xenobiotic amines. The cellular localization of these isoenzymes in the central nervous system (CNS) differs markedly and only partly reflects the distribution of their presumed natural substrates. In the present study, by using in situ hybridization with 35S-labelled oligonucleotide probes, we examined the distribution of mRNAs encoding MAO-A and MAO-B in the rat CNS. Probes for tyrosine hydroxylase, histidine decarboxylase, and tryptophan hydroxylase mRNAs were used to demonstrate the catecholaminergic, histaminergic, or serotoninergic nature of some cell populations in adjacent sections. The radioligands [3H]-Ro 41-1049 and [3H]lazabemide (reversible and selective inhibitors of MAO-A and MAO-B, respectively) were used to reveal the protein distribution by enzyme radioautography. The distribution and abundance of transcripts for both isoenzymes in the tissues investigated differed markedly but, in general, correlated with the protein distribution. MAO-A mRNA and protein were most abundant in noradrenergic neurons. However, moderate levels of transcript expression and protein were also detected in the serotoninergic neurons, and low but significant levels were detected in the dopaminergic neurons. An unexpectedly remarkable degree of hybridization signal was apparent in nonaminergic cell populations, e.g., in the cerebral cortices, the hippocampal formation (CA1-3, dentate gyrus), the cerebellar granule cell layer, and the spinal cord motoneurons. In contrast, MAO-B mRNA and protein were most abundant in serotoninergic and histaminergic neurons, Bergmann glial cells, and circumventricular organs, including the ependyma. MAO-B transcripts were also weakly expressed in nonaminergic cells, e.g., in the hippocampal formation (CA1-2). A further nonneuronal localization of MAO-B transcripts was also resolved, e.g., in the glia limitans, the olfactory nerve layer, and the cerebellar peduncle. These findings reveal further the potential of various cell populations to synthesize the isoenzymes, and homologous (aminergic) and heterologous (nonaminergic) patterns of expression as well as coexpression of MAO mRNAs are described.

The neurotoxm I-methyl-4-phenyl-1,2,3,6-tetrahydropyrldlne (MPTP)