Chromosomal localization and catalytic properties of the recombinant alpha subunit of human lymphocyte methionine adenosyltransferase.
Human lymphocyte methionine adenosyltransferase (HuLy MAT) consists of heterologous subunits alpha and beta. The cDNA sequence of the alpha subunit of HuLy MAT from Jurkat leukemic T cells was identical to that of the human kidney alpha subunit and highly homologous to the sequence of the extrahepatic MAT from other sources. The 3'-untranslated sequence was found to be highly conserved, suggesting that it may be important in regulating the expression of MAT. The extrahepatic alpha subunit unit of MAT was found to be expressed also in human liver, and no differences were found in the sequence of the alpha subunit from normal and malignant T cells. The sequence of two unspliced introns found in the cDNA clones from the Jurkat library enabled us to isolate genomic clones harboring the human extrahepatic alpha subunit gene and to localize it to the centromere on chromosome arm 2p, an area that corresponds to band 2p11.2. Expression of the alpha subunit cDNA in Escherichia coli yielded two peptides with the immunoreactivity and mobilities of authentic alpha/alpha' subunits from HuLy. The Km of the recombinant alpha subunit was 80 microM, which is 20-fold higher than found for the (alpha alpha')x beta y holoenzyme purified from leukemic lymphocytes and 4-10-fold higher than found for the normal lymphocyte enzyme. The data suggest that the alpha/alpha' subunits mediate the enzyme catalytic activity and that the beta subunit may be a regulatory subunit of extrahepatic MAT.
De La Rosa
J
J
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Ostrowski
J
J
Hryniewicz
M M
MM
Kredich
N M
NM
Kotb
M
M
LeGros
H L
HL
Valentine
M
M
Geller
A M
AM
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002871:Chromosome Banding; D002874:Chromosome Mapping; D002889:Chromosomes, Human, Pair 2; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D015723:Gene Library; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008214:Lymphocytes / Q000201:enzymology*; D046911:Macromolecular Substances; D008716:Methionine Adenosyltransferase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000201:enzymology; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D008716:Methionine Adenosyltransferase