Human kidney thiopurine methyltransferase (TPMT): Photoaffinity labeling with ( sup 3 H-methyl)- S-adenosyl-L-methionine ( sup 3 H-ado-met)
- 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-; CODEN: FAJOE
- Journal Information:
- FASEB Journal (Federation of American Societies for Experimental Biology); (United States), Vol. 4:3; Conference: 74. annual meeting of the Federation of American Societies for Experimental Biology, Washington, DC (United States), 1-5 Apr 1990; ISSN 0892-6638
- Country of Publication:
- United States
- Language:
- English
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METHYL TRANSFERASES
MEASURING METHODS
AMINO ACID SEQUENCE
AUTORADIOGRAPHY
DRUGS
ELECTROPHORESIS
ENZYME INHIBITORS
GENES
ISOENZYMES
KIDNEYS
MAN
METABOLISM
METHIONINE
PHOTOLYSIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
AMINO ACIDS
ANIMALS
BODY
CARBON-GROUP TRANSFERASES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DECOMPOSITION
ENZYMES
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
LIPOTROPIC FACTORS
MAMMALS
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PHOTOCHEMICAL REACTIONS
PRIMATES
PROTEINS
TRANSFERASES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques