Assessment of the relationship between genotypic status of a DT-diaphorase point mutation and enzymatic activity.
DT-diaphorase, a cytosolic reductase, has been implicated as an activator of chemotherapeutic prodrugs and a detoxifier of certain potentially carcinogenic xenobiotics. A common C to T nucleotide 609 substitution in DT-diaphorase cDNA has been associated with protein instability and reduced catalytic activity. The degree to which the allelic status of the substitution correlates with enzymatic activity was assessed in 45 normal human skin fibroblast strains using a PCR-RFLP assay. Included in this study was the 3437T strain, which is unique in that it is heterozygous for the polymorphism yet contains undetectable enzymatic activity. An allele-specific RT-PCR-RFLP technique attributed this phenomenon to exclusive DT-diaphorase mRNA expression from the variant allele. Overlap in activities was observed between individual strains homozygous for the wild-type allele and heterozygotes, but the former group displayed enzymatic activity that was on average 2-fold higher. Western blot analysis of the two strains in this panel that are homozygous for the variant allele revealed that they express relatively low amounts of DT-diaphorase protein, consistent with the role of the substitution in protein instability. This work confirms that genotypic status is a reliable initial estimate of DT-diaphorase activity.
Misra
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V
Department of Medical Biophysics, University of Toronto and Division of Experimental Therapeutics, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada.
Grondin
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A
Klamut
H J
HJ
Rauth
A M
AM
D000293:Adolescent; D000328:Adult; D002460:Cell Line; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D005838:Genotype; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D012867:Skin / Q000201:enzymology
D016660:NAD(P)H Dehydrogenase (Quinone)