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The Interaction of Dithiothreitol and Acetyl Coenzyme A in a Radiochemical Assay for Rat Brain ATP:Citrate Oxaloacetate Lyase
Author(s) -
Simpson J.
Publication year - 1981
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1981.tb05296.x
Subject(s) - citrate synthase , dithiothreitol , atp citrate lyase , biochemistry , chemistry , lyase , coenzyme a , enzyme , reductase
[ 14 C]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 a rapid, reproducible assay for rat brain ATP:citrate lyase using [1,5‐ 14 C]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 ≥79% by α‐ketoglutaric acid (10 m m ), Cu 2+ (1 m m )and Zn 2+ (1 m m ). [ 14 C]Oxaloacetate, [ 14 C]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.