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Ph regulation of mitochondrial branched chain alpha keto acid transport and oxidation in rat heart mitochondria

Journal of Biological Chemistry 262(20): 9629-9635

Ph regulation of mitochondrial branched chain alpha keto acid transport and oxidation in rat heart mitochondria

The kinetics of branched chain .alpha.-keto acid uptake and efflux were studied as a function of varied external and matrix pH. Matrix pH was determined by the distribution of 5,5'-dimethyloxazolidine-2,4-dione. When rat heart mitochondria were incubated under transport conditions at pH 7.0 with succinate as respiratory substrate, the matrix pH was significantly > 8.0. Matrix pH remained .gtoreq. 8.0 when the medium pH was varied from 6.3 to 8.3, and it was lowered below 8.0 by addition of 5 mM phosphate or uncoupler. No pH gradient was detectable when mitochondria were incubated in the presence of valinomycin and uncoupler. Efflux of .alpha.-ketoisocaproate or .alpha.-ketoisovalerate from rat heart mitochondria obeyed first order kinetics. Varying the external pH from 6.6 to 8.3 had no significant effect on efflux, and at an external pH of 7.0, the first order rate constant for efflux was not affected by decreasing the matrix pH. On the other hand, exchange was sensitive to changes in medium but not matrix pH. The K0.5 for external branched chain .alpha.-keto acid was lowered by changing the medium pH from 7.6 to 6.3. At medium pH values .gtoreq. 8.0 both K0.5 and Vmax were affected. Uptake was determined either by measuring initial rates or was calculated after measuring the first order approach to a final equilibrium value. Unlike efflux, uptake was sensitive to changes in both external and matrix pH. The rate of branched chain .alpha.-keto acid uptake was stimulated by decreasing the medium pH from 8.3 to 6.3 and by alkalinization of the mitochondrial matrix. The estimated external pK for proton binding was 6.9. The data inidcate that the branched chain .alpha.-keto acid transporter is asymmetric, that is, binding sites for substrate on the inside and outside of the mitochondrial membrane are not identical. .alpha.-Ketoisocaproate oxidation was measured at C in isolated mitochondria over the pH range of 6.6 to 8.1. Changes in the rate of branched chain .alpha.-keto acid oxidation, particularly when ATP was added (.uparw. .DELTA.pH), were found to parallel the pH effects observed on branched chain .alpha.-keto acid uptake. Therefore, transport, and by implication oxidation, can be regulated by pH changes within the physiological range. Furthermore, intracellular pH may affect the degree of compartmentation between the cytosolic and mitochondrial branched chain .alpha.-keto acid pools.

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Accession: 006092007

PMID: 3597428

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