Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria.
The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D005260:Female; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry
D007656:Ketoglutaric Acids; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D009249:NADP; D010088:Oxidoreductases; D007521:Isocitrate Dehydrogenase; D007655:Ketoglutarate Dehydrogenase Complex; D010749:Phosphoprotein Phosphatases; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase; D002118:Calcium