Stimulatory effect of calcium on metabolism and its sensitivity to pH in kidney mitochondria.
The relationship between mitochondrial matrix free Ca2+ concentration ([Ca2+]m) and pH was evaluated by incubating isolated rat kidney mitochondria with different extramitochondrial Ca2+ concentrations ([Ca2+]e) at medium pH (pHe) 7.0 and 7.4. [Ca2+]m was monitored using the fluorescent signal from mitochondria loaded with the Ca2+ indicator fura 2. The changes in [Ca2+]m were compared with alpha-ketoglutarate dehydrogenase (alpha-KGDH) flux, measured as O2 consumption (nmol.min-1.mg protein-1) from 185 microM alpha-ketoglutarate (alpha-KG). The apparent dissociation constant of the matrix fluorescent probe for Ca2+ was determined in each experiment and was 323 +/- 45 nM (n = 14). When mitochondria were exposed to [Ca2+]e below 160 nM, [Ca2+]m was greater at pHe 7.0 than at pHe 7.4. However, above 160 nM [Ca2+]e, [Ca2+]m plateaued at pHe 7.0 but rose progressively at pHe 7.4. Increasing [Ca2+]m by consecutive additions of Ca2+ to the medium had a significantly more pronounced acceleratory effect on alpha-KG oxidation at pHe 7.0 than at pHe 7.4. Kinetic analysis of alpha-KGDH revealed a 45% decrease in the Michaelis constant (Km) for alpha-KG at pHe 7.0, but the Km was unchanged at pHe 7.4 with elevation of [Ca2+]m from 32 to 751 nM. Maximal velocity (Vmax) increased significantly at both pHe values. Half-maximal alpha-KG oxidation occurred at [Ca2+]m of 76 +/- 11 nM and 105 +/- 31 nM at pHe 7.0 and 7.4, respectively. These studies demonstrate a direct, pH-sensitive correlation between [Ca2+]e and [Ca2+]m; [Ca2+]m changed over a range that may regulate alpha-KGDH flux in intact kidney mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)
Drewnowska
K
K
Department of Internal Medicine, Medical College of Virginia, Richmond 23298.
Schoolwerth
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AC
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D016257:Fura-2; D006863:Hydrogen-Ion Concentration; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D009994:Osmolar Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley
D007655:Ketoglutarate Dehydrogenase Complex; D002118:Calcium; D016257:Fura-2