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...
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.
A structure consisting of poly(A) complexed with other components is released from polysomes by ribonuclease treatment. The poly(A) complex has a sedimentation value of 12-15, while the corresponding sedimentation value for free poly(A) is 4. The complex does not appear to represent an artifact formed by interaction of free poly(A) with either cytoplasmic or polysomal proteins. The polynucleotide released from the complex by treatment with sodium dodecyl sulfate shows the same electrophoretic mobility as that of poly(A) isolated from deproteinized polysomal RNA. The poly(A) in the complex is partially protected from digestion by T2 ribonuclease. At least part of the poly(A) is available for base pairing with poly(U). The components associated with the poly(A) cause it to bind to Millipore filters at low ionic strength. These components are removed from the complex by Pronase digestion. The findings indicate that the poly(A) segment in messenger RNA serves as a binding site for a particle. This particle appears to consist of proteins.
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