Human DT-diaphorase, a potential cancer protecting enzyme. Its purification from abdominal adipose tissue.
The flavoprotein DT-diaphorase (EC 1.6.99.2) is believed to play an important role in the body's defense system. This enzyme has been purified 13,000-fold with a recovery of 58% from a cytosolic fraction of abdominal fat obtained from an obese patient undergoing elective surgery. Purification of the enzyme to electrophoretic homogeneity was achieved after two chromatographic steps: (1) affinity chromatography on azodicumarol Sepharose 6B; (2) anion exchange chromatography on DEAE Sephacel. The enzyme exhibits a monomer molecular mass of 32 kDa in SDS-PAGE and has 1 FAD prosthetic group per 32 kDa monomer. The FAD prosthetic group appears to be firmly attached to the apoproprotein. The enzyme reduces azodyes and quinones and demonstrates a broad substrate specificity. The enzyme has characteristics that are similar to DT-diaphorase purified from rodent liver, especially the rat liver enzyme. Estimated Km values for NADH, NADPH and menadione are 200, 140 and 3.3 microM, respectively. Vmax values for these substrates in the same order are 762, 667 and 294 mumol/mg.min. Dicumarol and warfarin exhibited competitive inhibition with pyridine nucleotides. The inhibition constants (Ki) for the drugs were estimated to be 10 nM and 2.2 microM, respectively. When compared to several other tissues, abdominal fat has one of the highest DT-diaphorase activities (Martin, L.F., Patrick, S.D. and Wallin, R. (1987) DT-diaphorase in morbidly obese patients. Cancer Lett., 36, 341-347), but the specific role of the enzyme in human fat is unknown.
Smith
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Department of Surgery, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Martin
L F
LF
Wallin
R
R
D000005:Abdomen / Q000201:enzymology*; D000273:Adipose Tissue / Q000201:enzymology*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D001728:Dicumarol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D016660:NAD(P)H Dehydrogenase (Quinone); D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D014859:Warfarin / Q000494:pharmacology
D014859:Warfarin; D001728:Dicumarol; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases