Purified and isolated Mat II &bgr; subunit nucleic acids and...

Details Purified and isolated Mat II &bgr; subunit nucleic acids and... Purified and isolated Mat II &bgr; subunit nucleic acids and...

2000-06-30

2004-02-24

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S006120, C435S252300, C435S320100, C435S325000, C435S410000, C536S023200

Reexamination Certificate

active

06696279

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to isolated and purified proteins and nucleic acids which modulate the biological activity of methionine adenosyltransferase II (MAT II). More particularly, the present invention relates to isolated and purified MAT II &bgr; subunit polypeptides and isolated and purified nucleic acids encoding the same.
Table of Abbreviations
BSA
bovine serum albumin
CDR(s)
complementarity determining region(s)
EST
expressed sequence tags
GC-MS
gas chromatography-Mass spectroscopy
HAT
cell culture media comprising hypoxanthine,
aminopterin, and thymidine
HBSS
Hank's balanced salt solution
HPLC
high pressure liquid chromatography
KLH
keyhole limpet hemocyanin
MAT II
methionine adenosyltransferase II
myc
human oncogene used herein as molecular tag
ORF
open reading frame
PCR
polymerase chain reaction
PBMC
peripheral blood mononuclear cells.
RACE
rapid amplification of cDNA ends
RNAi
RNA interference assay
UTR
untranslated region
BACKGROUND ART
Methionine adenosyltransferase (MAT; S-adenosyl-L-methionine (AdoMet) synthetase, EC 2.5.1.6) is an essential enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet) from L-methionine (L-Met) and ATP (Cantoni, G. L. (1953)
J. Biol. Chem
. 204: 403-416; Mudd, S. H. (1973) The Adenosyltransferases, Third Edition Ed. The Enzymes, Group Transfer (Part A) (Bayer, P. D., Ed.), III). AdoMet is the major methyl group donor, participating in the methylation of proteins, DNA, RNA, phospholipids and other small molecules (reviewed in Finkelstein et al. (1975)
Biochem. Biophys. Res
. Commun. 66: 81-7; Tabor, C. W., and Tabor, H. (1984)
Adv. Enzymol. Relat. Areas Mol. Biol
. 56: 251-82; Mudd et al. (1995) Disorders of transsulfuration, 7th Ed. The Molecular and Metabolic Basis of Inherited Diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.), McGraw-Hill Inc., New York). 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 (Finkelstein et al. (1975) Biochem. Biophys. Res. Commun. 66: 81-7; Tabor, C. W., and Tabor, H. (1984) Adv. Enzymol. Relat. Areas Mol. Biol. 56: 251-82; Mudd et al. (1995) Disorders of transsulfuration, 7th Ed. The Molecular and Metabolic Basis of Inherited Diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.), McGraw-Hill Inc., New York). MAT is present in all living species, including thermophilic archaebacteria, plants, yeast, and mammals (reviewed in Tabor, C. W., and Tabor, H. (1984) Adv. Enzymol. Relat. Areas Mol. Biol. 56: 251-82; Kotb, M., and Geller, A. M. (1993) Pharmacol. Ther. 59: 125-43; Chiang, et al. (1996) FASEB J. 10:471-80; Mato et al. (1997) Pharmacol. Ther. 73: 265-80). Interestingly, most species have more than one MAT isozyme (Kotb, M., and Geller, A. M. (1993) Pharmacol. Ther. 59: 125-43).
Mammalian MAT exists in multiple forms that differ in their physical and kinetic properties among distinct species and even among different tissues of the same species. In mammals there are three forms designated MAT I, II, and III that differ in their tissue distribution and kinetic properties (Hoffman, J. L. (1983) Methods Enzymol. 94, 223-8; Mato et al. (1994) Adv. Exp. Med. Biol. 368, 113-7; Okada et al. (1981) Biochemistry 20, 934-40; Kotb et al. (1997) Trends Genet. 13, 51-2). MAT I and III are referred to as the hepatic forms because their expression is confined to the liver. By contrast, MAT II is found in all mammalian tissues that have been examined to date, including erythrocytes, lymphocytes, brain, kidney, testis, and liver (Okada et al. (1981) Biochemistry 20, 934-40; Oden, K., and Clarke, S. (1983) Biochemistry 22, 2978-2986; Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30; Langkamp-Henken et al. (1994) Biochim. Biophys. Acta 1201, 397-404; Liau et al. (1979) Cancer Res. 39, 162-69; Sullivan, D. M., and Hoffman, J. L. (1983) Biochemistry 22, 1636-41; Mitsui et al. (1988) J. Biol. Chem. 263, 11211-16; Horikawa et al. (1990) J. Biol. Chem. 265, 13683-86).
MAT I is a tetramer and MAT III is a dimer of an identical catalytic subunit, &agr;1, encoded by the MATIA gene (Hoffman, J. L. (1983) Methods Enzymol. 94, 223-8; Horikawa, S., and Tsukada, K. (1991) Biochem. Int. 25, 81-90; Alvarez et al. (1993) Biochem. J. 293, 481-86; Sakata et al. (1993) J. Biol. Chem. 268, 13978-86; Ubagai et al. (1995) J. Clin. Invest. 96, 1943-47). On the other hand MAT II from leukemic T cells or from activated human lymphocytes is a hetero-oligmer consisting of &agr;2 (53 kDa), &agr;′2 (51 kDa) and &bgr; (38 kDa) subunits (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30). The &agr;2 and &agr;′2 are the catalytic subunits while &bgr; appeared to have a regulatory function (De La Rosa et al. (1992) J. Biol. Chem. 267, 10699-704; De La Rosa et al. (1995) J. Biol. Chem. 270:21860-68; LeGros et al. (1997) J. Biol. Chem. 272, 16040-47; LeGros et al. (1999) Submitted). The &agr;2 and &agr;′2 subunits are immunologically crossreactive and essentially identical to each other, but quite different from the &bgr; subunit. The &agr;2 subunit, which appears to be posttranslationally processed to yield &agr;′2 (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30), is encoded by the MAT2A gene which is homologous, but different from MAT1A gene (Kotb et al. (1997) Trends Genet. 13, 51-2; Horikawa, S., and Tsukada, K. (1991) Biochem. Int. 25, 81-90; De La Rosa et al. (1995) J. Biol. Chem. 270:21860-68).
The human MAT II from human lymphocytes has been analyzed to a certain extent (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260: 3923-30; Kotb, M., and Kredich, N. M. (1990) Biochim. Biophys. Acta 1039(2): 253-60; De La Rosa et al. (1992) J. Biol. Chem. 267: 10699-704; De La Rosa et al. (1995) J. Biol. Chem. 270, 21860-8; LeGros et al. (1997) J. Biol. Chem. 272, 16040-7), and it has been shown that the form present in activated lymphocytes consists of distinct subunits (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30; De La Rosa et al. (1992) J. Biol. Chem. 267, 10699-704). The catalytic MAT II &agr;2 subunit, which is encoded by the MAT2A gene, was cloned and characterized and found to be homologous, but different from the catalytic &agr;1 subunit of the liver MAT I/III isozyme (Horikawa, S., and Tsukada, K. (1991) Biochem. Int. 25, 81-90; Alvarez et al. (1993) Biochem. J. 293, 481-6; Sakata et al. (1993) J. Biol. Chem. 268, 13978-86; Horikawa et al. (1990) J. Biol. Chem. 265, 13683-6; De La Rosa et al. (1995) J. Biol. Chem. 270, 21860-8). The MAT II &agr;2 subunit, which has a calculated molecular weight of 43,600, migrates on SDS-PAGE gels as a 53 kDa protein, and is postranslationally modified to generate MAT II &agr;2′ subunit (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30). The catalytic &agr;2/&agr;2′ subunits are found in native MAT II associated with a catalytically inactive subunit designated MAT II &bgr;, which migrates on SDS-PAGE as a 38-kDa protein (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30; De La Rosa et al. (1992) J. Biol. Chem. 267, 10699-704; LeGros et al. (1997) J. Biol. Chem. 272, 16040-7).
Earlier work has shown that physiological activation of human lymphocytes induces downregulation of the &bgr; subunit with co-incidental alterations in MAT II kinetic properties (LeGros et al. (1997) J. Biol. Chem. 272, 16040-47). However, this differential expression of the &bgr; subunit has not been fully characterized. Further characterization of the MAT II &bgr; subunit, including the regulatory role of the MAT II &bgr; subunit, thus represents an ongoing need in the art.
SUMMARY OF THE INVENTION
The present invention discloses isolated and purified nucleic acids encoding the subunit of methionine adenosyltransferase II (MAT II), to isolated and purified MAT II &bgr; subunit polypeptides, and to the characterization of the role played by the MAT II &bgr; subunit in modulati

Affiliated with

Also associated with

Rating

Purified and isolated Mat II &bgr; subunit nucleic acids and... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Purified and isolated Mat II &bgr; subunit nucleic acids and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Purified and isolated Mat II &bgr; subunit nucleic acids and... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3330388