MECHANISM OF THE ACCELERATION OF CO2 PRODUCTION FROM PYRUVATE IN LIVER-MITOCHONDRIA BY HCO3(-)

To investigate the mechanism by which HCO3- accelerates pyruvate metab olism in guinea pig liver mitochondria, we measured continuously, at p H 7.4 and 37 degrees C, (CO2)-C-13-O-16 production from [1-C-13]pyruva te by mass spectrometry and NADH concentration by fluorescence and ana lyzed total malate, citrate, and beta-hydroxybutyrate produced by stan dard biochemical methods. When [1-C-13]pyruvate is added to the mitoch ondrial suspension, (CO2)-C-13-O-16 concentration rises steeply in the first seconds and then slows to a steady lower rate. Carbonic anhydra se (CA) eliminates this initial phase, which shows that decarboxylatio n of pyruvate produces CO2, not HCO3-, and it does this more rapidly t han it can equilibrate without CA. HCO3- (25 mM) increased (CO2)-C-13- O-16 production, O-2 consumption, and total malate and citrate product ion and decreased NADH concentration and total beta-hydroxybutyrate pr oduction. After obtaining the total amount of (CO2)-C-13-O-16, malate, citrate, and beta-hydroxybutyrate produced, we calculated that the ad dition of 25 mM HCO3- to the suspension medium increased the amount of pyruvate decarboxylated by pyruvate dehydrogenase (PDH) 16% and incre ased the amount carboxylated by pyruvate carboxylase 300%. This suppor ts our initial proposal that HCO, accelerates the pyruvate carboxylati on, which in turn consumes ATP directly and NADH and acetyl CoA second arily, all of which increase PDH activity. However, we found no accele ration of pyruvate decarboxylation by 0.5 and 1 mu M free Ca2+ concent ration, unless the mitochondria were uncoupled and ATP was added.