Baculovirus-mediated high level expression of a human thiopurine methyl transferase.
We have expressed the human thiopurine methyltransferase cDNA in a baculovirus vector in Sf21 (Spodoptera frugiperda) cells. This system expresses the enzyme at levels such that the thiopurine methyltransferase enzyme may be readily visualised by Coomassie blue stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The expressed enzyme catalysed the methylation of 6-mercaptopurine with an apparent Km of 892 microM, similar to that observed in human liver cytosol ie. 657 microM however, the Vmax was 13,500 pmole/mg/min, which is approximately 400 times higher than the Vmax observed in human liver cytosol ie. 33 pmole/mg/min. The thiopurine methyltransferase inhibitors 6-thioxanthine, p-methoxybenzoic acid and 3,5-dimethoxy benzoic acid were found to be potent inhibitors of the expressed enzyme.
Jackson
H
H
School of Pharmacy and Medical Sciences, University of South Australia, Adela
Hughes
B P
BP
Stupans
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I
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D015122:Mercaptopurine / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D015122:Mercaptopurine; D008780:Methyltransferases; C022745:thiopurine methyltransferase