The interaction of dithiothreitol and acetyl coenzyme A in a radiochemical assay for rat brain ATP:citrate oxaloacetate lyase.
[14C]Acetyl-CoA was found to react spontaneously with dithiothreitol to give a relatively apolar product which was readily extractable into a butanol-toluene scintillant. This technique was used in rapid, reproducible assay for rat brain ATP:citrate lyase using [1,5-14C]citrate as substrate. The tissue extract, a 14,000 g supernatant, exhibited a lyase activity of approximately 7 nmol acetyl-CoA produced/min per mg supernatant protein, and was inhibited greater than or equal to 79% by alpha-ketoglutaric acid (10 mM), Cu2+ (1 mM) and Zn2+ (1 mM). [14C]Oxaloacetate, [14C]malate and endogenous citrate synthase were found not to interfere significantly with lyase estimations, but NADH was required in the reaction mixture to inhibit acetyl-CoA hydrolase activity.
D001275:ATP Citrate (pro-S)-Lyase / Q000378:metabolism*; D000105:Acetyl Coenzyme A; D000818:Animals; D001921:Brain / Q000201:enzymology*; D004229:Dithiothreitol; D007700:Kinetics; D008297:Male; D009243:NAD; D051381:Rats
D009243:NAD; D000105:Acetyl Coenzyme A; D001275:ATP Citrate (pro-S)-Lyase; D004229:Dithiothreitol