Interaction of purified nicotinamidenucleotide transhydrogenase with dicyclohexylcarbodiimide.
The inhibition of the energy-linked nicotinamidenucleotide transhydrogenase (TH; EC 1.6.1.1) by dicyclohexylcarbodiimide (DCCD) has been further studied because of its important mechanistic implications. We had shown earlier that TH bound to submitochondrial particles from bovine heart is inhibited by DCCD and that NAD(H) protects the enzyme against this inhibition [Phelps, D.C., & Hatefi, Y. (1981) J. Biol. Chem. 256, 8217-8221]. By contrast, Pennington and Fisher [Pennington, R.M., & Fisher, R.R. (1981) J. Biol. Chem. 256, 8963-8969] working with purified TH concluded that NAD(H) does not protect against DCCD inhibition and that DCCD inhibition involves the TH proton channel rather than the nucleotide-binding active site. The present study shows that NAD(H) as well as AMP and ADP, which are known to bind to the NAD(H) binding site from competitive inhibition studies, protect the purified TH against inhibition by DCCD, whereas 2'-AMP and 3'-AMP, which bind to the NADP(H) site on TH, do not protect. In addition, it is shown that whereas the unmodified TH binds to NAD-agarose such that it is elutable by buffer containing NADH, the DCCD-modified enzyme does not bind to NAD-agarose. These results suggest strongly that DCCD binds at or near the NAD(H) binding site on TH. Another less likely possibility is that NAD(H) and DCCD bind to separate sites, but their bindings are mutually exclusive. With the use of [14C]DCCD, it has been shown that 100% activity inhibition corresponds to 0.5 mol of DCCD binding per mol of TH (Mr approximately 11 X 10(4].(ABSTRACT TRUNCATED AT 250 WORDS)
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D002234:Carbodiimides / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008929:Mitochondria, Heart / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000378:metabolism*
D002234:Carbodiimides; D009243:NAD; D000249:Adenosine Monophosphate; D009249:NADP; D004024:Dicyclohexylcarbodiimide; D000244:Adenosine Diphosphate; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases