Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity.
ATP-citrate lyase (CL) catalyzes the conversion of citrate and CoA to oxaloacetate (OA) and acetyl-CoA. As the coupled malic dehydrogenase (MDH) assay is not able either to study the effect of oxaloacetate (OA) on CL activity or to measure accurately CL activity in biological samples, a new assay was developed. The CL-citrate coupled CAT assay measures the amount of acetyl-CoA formed by transferring radiolabeled acetyl-CoA synthesized from [14C]citrate to chloramphenicol with chloramphenicol acetyltransferase (CAT). Employing this assay, the rate of increase in acetyl-CoA synthesis from citrate is linear with respect to added CL. Kinetic values for ATP, CoA and citrate are similar to those obtained using the MDH assay. The effect of CL phosphorylation on enzyme activity was determined. CL phosphorylated by cAMP-dependent protein kinase or by this kinase and glycogen synthase kinase-3 (GSK-3) decreases the apparent Vmax without changing the apparent Km. The effect of OA, a product of the enzyme reaction, on CL activity was also determined. Computational analysis of the data obtained without added OA and at three concentrations of OA indicate that the apparent Km for the substrate is not altered even though the apparent Vmax is decreased. The effect of OA on the activity of phosphorylated enzyme was also determined. OA decreases the apparent Vmax of the phosphorylated enzyme to the same extent as in control CL. This assay is able to measure CL activity in cytosol from 3T3-L1 adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Pentyala
S N
SN
Department of Physiology and Biophysics, State University of New York at Stony Brook 11794-8661, USA.
Benjamin
W B
WB
D016475:3T3 Cells; D001275:ATP Citrate (pro-S)-Lyase / Q000378:metabolism*; D000273:Adipose Tissue / Q000201:enzymology; D000818:Animals; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D010071:Oxaloacetates / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010766:Phosphorylation; D051381:Rats
D010071:Oxaloacetates; D001275:ATP Citrate (pro-S)-Lyase