Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase

Kotb, Malak; Geller, Arthur M.; Markham, George D.; Kredich, Nicholas M.; Rosa, James De La; Beachey, Edwin H.

doi:10.1016/0167-4838(90)90068-q

Cited by 18 publications

(16 citation statements)

References 23 publications

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“…4). In concordance with our previous studies (33), there was no immunological crossreactivity between the MAT II ␣ and ␤ subunits. Furthermore, no reactivity was detected between the anti-␤ peptides and protein extracts of E. coli host cells that were either untransfected or that were transfected with the same vector and expressing the human recombinant MAT II a 2 subunit cDNA (Fig.…”

Section: Cdna and Predicted Protein Sequence Of The Human Mat IIsupporting

confidence: 79%

“…By contrast, the ␤ subunit migrated on SDS-PAGE as a 38-kDa protein and had a peptide banding pattern that was quite distinct from that of the two ␣ 2 subunits, suggesting that ␤ is distinct from the ␣ 2 subunit. Antibodies to the pure MAT II enzyme from human leukemic cells, whose purity was verified by analytical ultracentrifugational analysis, recognized both ␣ 2 and ␤ subunits in human lymphocytes and showed cross-reactivity with E. coli and yeast MAT ␣ subunits, which is not surprising given the now known high degree of primary sequence homology of MAT catalytic subunits in the many species analyzed; however, there was no indication that the anti-human MAT II antibodies were recognizing a protein similar to the ␤ subunit in either E. coli or yeast cell extracts (33). Moreover, polyclonal antibodies to E. coli or yeast MAT reacted with the human MAT I, II, and III ␣ subunits but failed to recognize the MAT II ␤ subunit protein.…”

Section: Discussionmentioning

confidence: 92%

“…After electroblotting the proteins onto the nitrocellulose for 1 h at 25-30 V/cm, the blots were blocked overnight with 6% nonfat dry milk in TBS (50 mM Tris, pH 7.5, and 150 mM NaCl). Following the removal of the blocking solution, the blot was washed in TBS and incubated with primary polyclonal anti-holoenzyme antisera (33) or polyclonal antisera generated either against pure human recombinant MAT II ␣2 or ␤ subunits or against the two synthetic peptides copying sequences of the ␤ subunits. The blots were developed with secondary anti-rabbit antibodies conjugated to horseradish peroxidase and the luminol-chemiluminescence reagents (ECL; Amersham Pharmacia Biotech) as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

“…The catalytic ␣ 2 /␣ 2Ј subunits are found in native MAT II associated with a catalytically inactive subunit designated MAT II ␤, which migrates on SDS-PAGE as a 38-kDa protein (22,29,31). Inasmuch as the MAT II ␤ subunit had no homology to the ␣ 2 subunit (22,33), it was postulated that it is encoded by a different gene, which was putatively designated MAT2B (12). In this study, we report the complete sequence of cDNA encoding the entire MAT II ␤ subunit, and we show that the protein expressed in Escherichia coli associates with the E. coli as well as the human catalytic ␣ subunits of MAT.…”

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confidence: 99%

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Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

Legros¹,

Halim²,

Geller³

et al. 2000

Journal of Biological Chemistry

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MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic ␣ 2 /␣ 2 subunits and a noncatalytic ␤ subunit, believed to have a regulatory function. The full-length cDNA that encodes the ␤ subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The ␤ subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native ␤ subunit protein, and to the r␤ protein. There is no cross-reactivity between the MAT II ␣ 2 or ␤ subunits. None of the anti-␤ subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no ␤ subunit protein. Interestingly, the r␤ subunit associated with E. coli as well as human MAT ␣ subunits. This association changed the kinetic properties of both enzymes and lowered the K m of MAT for L-methionine. Together, the data show that we have cloned and expressed the human MAT II ␤ subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells.Methionine adenosyltransferase (MAT; S-adenosyl-L-methionine synthetase, EC 2.5.1.6) 1 is an essential enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet) from L-methionine (L-Met) and ATP (1, 2). AdoMet is the major methyl group donor, participating in the methylation of proteins, DNA, RNA, phospholipids, and other small molecules (reviewed in Refs. 3-5). In addition, AdoMet is the ultimate source of the propylamine moiety used in polyamine biosynthesis, and it serves as co-factor for other key enzymes in the one-carbon metabolism pathway (3-5). MAT is present in all living species, including thermophilic archaebacteria, plants, yeast, and mammals (reviewed in Refs. 4 and 6 -8). Interestingly, most species have more than one MAT isozyme (6).In mammals, it is now established that there are at least two MAT isozymes (9 -12). MAT I/III is expressed only in liver and has a catalytic subunit designated ␣ 1 that is encoded by the MAT1A gene (8, 9, 13-16). MAT I and MAT III represent different oligomeric forms of the ␣ 1 subunit -MAT III is a dimer, and MAT I is a tetramer of the ␣ 1 subunit (9, 17-19). MAT I and MAT III differ considerably in their physical, kinetic, and regulatory properties (8,9,20). The MAT II isozyme is expressed in all tissues, including the liver, and has been studied in many tissues including erythrocytes, lymphocytes, brain, kidney, testis, and fetal liver (11, 20 -27).We have been characterizing the human MAT II from human lymphocytes (22, 28 -31) 2 and were able to show that the form present in activated lymphocytes consists of distinct subunits (22...

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Section: Cdna and Predicted Protein Sequence Of The Human Mat IIsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 92%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

Legros¹,

Halim²,

Geller³

et al. 2000

Journal of Biological Chemistry

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“…The ␤ subunit lowers the K m value of the enzyme for L-Met and confers susceptibility to product inhibition by AdoMet (26,31). Interestingly, we have not been able to detect the ␤ protein in E. coli or yeast extracts (26,35) despite the presence of a high level of homology between this protein and enzymes that catalyze the reduction of thiamine diphosphate-linked sugars in bacteria (36). Therefore, the unique role and significance of the ␤ protein in mammalian cells represents an intriguing area of study, particularly because ␤ is differentially expressed in normal and leukemic T cells and subsequently affects AdoMet levels.…”

Section: Discussionmentioning

confidence: 71%

Regulation of the Human MAT2B Gene Encoding the Regulatory β Subunit of Methionine Adenosyltransferase, MAT II

LeGros

Halim

Chamberlin³

et al. 2001

Journal of Biological Chemistry

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Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet), a key molecule in transmethylation reactions and polyamine biosynthesis. The MAT II isozyme consists of a catalytic ␣2 and a regulatory ␤ subunit. Down-regulation of the MAT II ␤ subunit expression causes a 6 -10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the ␤ subunit expression is regulated, we cloned the MAT2B gene, determined its organization, characterized its 5-flanking sequences, and elucidated the in vitro and in vivo regulation of its promoter. Transcription of the MAT2B gene initiates at position ؊203 relative to the translation start site. Promoter deletion analysis defined a minimal promoter between positions ؉52 and ؉93 base pairs, a GC-rich region. Inclusion of the sequences between ؊4 and ؉52 enhanced promoter activity; this was primarily because of an Sp1 recognition site at ؉9/؉15. The inclusion of sequences up to position ؊115 provided full activity; this was attributed to a TATA at ؊32. The Sp1 site at position ؉9 was key for the formation of protein⅐DNA complexes. Mutation of both the Sp1 site at ؉9 and the TATA at ؊32 reduced promoter activity to its minimal level. Supershift assays showed no effect of the anti-Sp1 antibody on complex formation, whereas the anti-Sp3 antibody had a strong effect on protein⅐DNA complex formation, suggesting that Sp3 is one of the main factors binding to this Sp1 site. Chromatin immunoprecipitation assays supported the involvement of both Sp1 and Sp3 in complexes formed on the MAT2B promoter. The data show that the 5-untranslated sequences play an important role in regulating the MAT2B gene and identifies the Sp1 site at ؉9 as a potential target for modulating MAT2B expression, a process that can have a major effect on intracellular AdoMet levels. Methionine adenosyltransferase (MAT)1 (ATP⅐L-methionine S-adenosyltransferase) (EC 2.5.1.6) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet) (1, 2). AdoMet is the major methyl donor in transmethylation reactions and the propylamine donor in the biosynthesis of polyamines (3-5). Furthermore, AdoMet participates as a co-factor in key metabolic pathways (3-5). Most species studied to date have more than one MAT isozyme (6). In mammals, the two major MAT isozymes are designated MAT I/III and MAT II (7-10). The MAT I/III isozymes are, respectively, a tetramer and dimer of a catalytic ␣1 subunit that is expressed only in liver (7, 11-13). The MAT II isozyme is expressed in all tissues including the liver (9, 14 -21). Previous studies from our group (15, 22) have determined that MAT II from human leukemic T and B cells, as well from activated human lymphocytes, is a hetero-oligomer that consists of ␣2 (53 kDa), ␣2Ј (51 kDa), and ␤ (38 kDa) subunits. The ␣2/␤ hetero-oligomeric composition of MAT II was also determined in bovine brain, Ehrlich ascites tumor, and calf thymus (15,19). The ␣2 subunit is responsible for the enzyme catalytic activity and is post-translationally modifie...

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Inhibition of Methionine Adenosyltransferase by the Polyamines

Geller

Legros

Wherry

et al. 1997

Archives of Biochemistry and Biophysics

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Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase

Cited by 18 publications

References 23 publications

Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

Regulation of the Human MAT2B Gene Encoding the Regulatory β Subunit of Methionine Adenosyltransferase, MAT II

Inhibition of Methionine Adenosyltransferase by the Polyamines

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