Catechol O-methyltransferase pharmacogenetics: photoaffinity labelling and western blot analysis of human liver samples.
The level of catechol O-methyltransferase (COMT) activity and COMT thermal stability in human tissue are controlled by a common genetic polymorphism. We studied individual hepatic biopsy samples shown previously to have phenotypically high, low or intermediate COMT activities and thermal stabilities to test the hypothesis that the molecular mass (M(r)) and/or isoelectric point (pI) of the enzyme might differ in tissue from subjects with different presumed genotypes for the COMT genetic polymorphism. COMT was partially purified from each hepatic tissue sample by sequential ion exchange and gel filtration chromatography, and photoaffinity labelling was performed with [3H-methyl]-S-adenosyl-L-methionine ([3H-methyl]-Ado-Met), the methyl donor for the COMT enzymatic reaction. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) analysis of individual samples consistently showed the presence of three [3H-methyl]-Ado-Met photoaffinity labelled proteins with pI values of 5.4, 5.5 and 5.7, all three of which had M(r) values of approximately 27.1 kDa. The same pattern was observed in all samples irrespective of COMT phenotype. Western blot analysis of 2-D SDS-PAGE gels performed with rabbit polyclonal antibodies to partially purified human kidney COMT showed a pattern similar to that found during photoaffinity labelling. Once again, the same pattern was found in all samples irrespective of COMT phenotype. Therefore, neither photoaffinity labelling nor Western blot analysis revealed differences in either M(r) or pI of cytoplasmic COMT in hepatic tissue from subjects selected on the basis of different phenotypic expression of the COMT genetic polymorphism.
Department of Pharmacology, Mayo Clinic/Mayo Foundation, Rochester, MN 55905.
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S
Aksoy
J
J
Klener
R M
RM
Weinshilboum
D000345:Affinity Labels; D015153:Blotting, Western; D002394:Catechol O-Methyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D006801:Humans; D008099:Liver / Q000201:enzymology*; D011110:Polymorphism, Genetic; D012436:S-Adenosylmethionine / Q000378:metabolism
D000345:Affinity Labels; D012436:S-Adenosylmethionine; D002394:Catechol O-Methyltransferase