THE FORMATION OF ACETOACETATE FROM ACETYL COENZYME A, MALONYL COENZYME A, AND PYRUVATE BY WASHED MITOCHONDRIA ISOLATED FROM GUINEA PIG LIVER

Washed mitochondria isolated from guinea pig liver were capable of synthesizing acetoacetate from pyruvate. Both acetyl-CoA and malonyl-CoA were incorporated into acetoacetate in the presence of pyruvate. However, without pyruvate, only acetyl-CoA was incorporated to any significant extent. Tracer experiments indicated that although malonyl-CoA was incorporated into acetoacetate, increased acetoacetate synthesis in the presence of pyruvate plus malonyl-CoA resulted primarily from increased pyruvate incorporation.The results of the present experiments indicated that a CO2fixation step was involved in the conversion of pyruvate to acetoacetate. Evidence in favor of this was based on the inhibitory effects of avidin (with partial reversal by biotin), stimulation with increasing bicarbonate concentration, and increased acetoacetate synthesis in the presence of malonyl-CoA.Sulphydryl binding agents completely inhibited acetoacetate synthesis from pyruvate. In the formation of acetoacetate, carbon atom 1 of pyruvate was eliminated. This indicated that pyruvate was converted into an active 2-carbon unit.