Human kidney thiopurine methyltransferase (TPMT): Photoaffinity labeling with ( sup 3 H-methyl)- S-adenosyl-L-methionine ( sup 3 H-ado-met)
Conference
·
· FASEB Journal (Federation of American Societies for Experimental Biology); (United States)
OSTI ID:5715227
- Mayo Clinic/Mayo Foundation, Rochester, MN (United States)
TPMT (EC 2.1.1.67) catalyzes the S-methylation of 6-mercaptopurine (6-MP) and other heterocyclic and aromatic thiol drugs. TPMT activity and immunoreactive protein in humans are regulated by a common genetic polymorphism. Human kidney contains two isozymes of TPMT which can be separated by ion exchange chromatography. Partially purified isozymes of human kidney TPMT were exposed to UV light in the presence of {sup 3}H-Ado-Met. After photolysis, these preparations were subjected to SDS-polyacrylamide gel electrophoresis. Autoradiography of gels revealed that a 34 kDa protein was the predominant species that was radioactively labeled for both isozymes. Photoaffinity labeling of TPMT was inhibited in a concentration-dependent fashion by the methyl acceptor substrate, 6-MP, and by three TPMT inhibitors, 3,4-dimethoxy-5-hydroxybenzoic acid, 6-methylmercaptopurine and S-adenosyl-L-homocysteine. Of three wavelengths tested, 254, 300, and 350 nm, labeling was the greatest at 254 nm, near the absorption maximum for Ado-Met. Photoaffinity labeling of TPMT with {sup 3}H-Ado-Met should make it possible to determine the amino acid sequence of the active site and may make it possible to define the molecular basis of the genetic polymorphism for this important drug-metabolizing enzyme.
- OSTI ID:
- 5715227
- Report Number(s):
- CONF-9004153--
- Conference Information:
- Journal Name: FASEB Journal (Federation of American Societies for Experimental Biology); (United States) Journal Volume: 4:3
- Country of Publication:
- United States
- Language:
- English
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· Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
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Tue Sep 23 00:00:00 EDT 2008
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·
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OSTI ID:285049
Related Subjects
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
AMINO ACIDS
ANIMALS
AUTORADIOGRAPHY
BODY
CARBON-GROUP TRANSFERASES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DECOMPOSITION
DRUGS
ELECTROPHORESIS
ENZYME INHIBITORS
ENZYMES
GENES
HYDROGEN COMPOUNDS
ISOENZYMES
ISOTOPE APPLICATIONS
KIDNEYS
LIPOTROPIC FACTORS
MAMMALS
MAN
MEASURING METHODS
METABOLISM
METHIONINE
METHYL TRANSFERASES
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
PRIMATES
PROTEINS
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
VERTEBRATES
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
AMINO ACIDS
ANIMALS
AUTORADIOGRAPHY
BODY
CARBON-GROUP TRANSFERASES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DECOMPOSITION
DRUGS
ELECTROPHORESIS
ENZYME INHIBITORS
ENZYMES
GENES
HYDROGEN COMPOUNDS
ISOENZYMES
ISOTOPE APPLICATIONS
KIDNEYS
LIPOTROPIC FACTORS
MAMMALS
MAN
MEASURING METHODS
METABOLISM
METHIONINE
METHYL TRANSFERASES
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
PRIMATES
PROTEINS
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
VERTEBRATES