Mechanism of the acceleration of CO2 production from pyruvate in liver mitochondria by HCO3-.
To investigate the mechanism by which HCO3- accelerates pyruvate metabolism in guinea pig liver mitochondria, we measured continuously, at pH 7.4 and 37 degrees C, 13C16O2 production from [1-13C]pyruvate by mass spectrometry and NADH concentration by fluorescence and analyzed total malate, citrate, and beta-hydroxybutyrate produced by standard biochemical methods. When [1-13C]pyruvate is added to the mitochondrial suspension, 13C16O2 concentration rises steeply in the first seconds and then slows to a steady lower rate. Carbonic anhydrase (CA) eliminates this initial phase, which shows that decarboxylation of pyruvate produces CO2, not HCO3-, and it does this more rapidly than it can equilibrate without CA. HCO3- (25 mM) increased 13C16O2 production, O2 consumption and total malate and citrate production and decreased NADH concentration and total beta-hydroxybutyrate production. After obtaining the total amount of 13C16O2, malate, citrate, and beta-hydroxybutyrate produced, we calculated that the addition of 25 mM HCO3- to the suspension medium increased the amount of pyruvate decarboxylated by pyruvate dehydrogenase (PDH) 16% and increased the amount carboxylated by pyruvate carboxylase 300%. This supports our initial proposal that HCO3- accelerates the pyruvate carboxylation, which in turn consumes ATP directly and NADH and acetyl CoA secondarily, all of which increase PDH activity. However, we found no acceleration of pyruvate decarboxylation by 0.5 and 1 microM free Ca2+ concentration, unless the mitochondria were uncoupled and ATP was added.
Taguchi
Y
Y
Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, 19104, USA.
Ono
Y
Y
Lin
L
L
Storey
B T
BT
Dodgson
S J
SJ
Forster
R E
RE
D020155:3-Hydroxybutyric Acid; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology*; D002245:Carbon Dioxide / Q000378:metabolism*; D002247:Carbon Isotopes; D002256:Carbonic Anhydrases / Q000494:pharmacology; D002417:Cattle; D002951:Citrates / Q000378:metabolism; D006168:Guinea Pigs; D006885:Hydroxybutyrates / Q000378:metabolism; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D011766:Pyruvate Carboxylase / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism*
D001639:Bicarbonates; D002247:Carbon Isotopes; D002951:Citrates; D006885:Hydroxybutyrates; D008293:Malates; D011768:Pyruvate Dehydrogenase Complex; D011773:Pyruvates; D009243:NAD; D002245:Carbon Dioxide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002256:Carbonic Anhydrases; D011766:Pyruvate Carboxylase; D020155:3-Hydroxybutyric Acid