Activation of Endogenous Respiration and Anion Transport in Corn Mitochondria by Acidification of the Medium

Abstract

Acdification of the suspending medium of corn mitocbondrina (Zea mays L., WF9 x Mol7) from pH 7.5 to pH 6.8 to 6.4 It has been reported (6) that corn mitochondria show pronounced contraction or shrinkage as judged by light transmission when the medium is acidified. There were indications that certain anions, such as citrate or acetate, opposed the acid contraction, suggesting salt uptake driven by the pH gradient. However, in terms of Mitchell's chemiosmotic hypothesis (9) this should not happen. Net salt uptake requires a proton motive force (Ap = Ai -59ApH, mV at 25 C) with At# driving cation uptake as an electrophoretic uniport, and ApH driving anion uptake as a neutral symport or antiport. Simply acidifying the medium does not provide the requisite electrical potential.We have investigated this problem using sulfate, phosphate, and citrate as transportable anions. Acidification of the medium by about 1 pH unit does produce osmotic swelling, but the transport is primarily due to activation of endogenous respiration. MATERIALS AND METHODSMitochondria were isolated from corn shoots (Zea mays L. WF9 x Mol7) essentially as previously described (6), but with the isolation medium changed to 0.4 M sucrose, 50 mm TES buffer, 1 mg/ml of BSA, 3 mm EDTA, 3 mM MgCl2, adjusted to pH 7.6 with KOH. Final suspension was in 0.25 M sucrose plus 1 mg/ml of BSA.Measurements of per cent transmission, pH, and 02 were as previously described (6) but at room temperature (23 C). Full scale transmittance was set from 20 to 40%, adjusting slit width to bring initial transmittance to 30%. Cuvettes contained 4 ml of aerated 100 mm sucrose, 10 mm TES, 1 mg/ml of BSA, adjusted to pH 7.5; however, after addition of 0.7 to 1 mg of ' Supported by United States Energy Research and DevelopmentAdministration Grant E-(11-1)-790. mitochondrial protein the actual pH recorded was sometimes as low as 7.4. Additions of acids and salts to the medium were from 1 M stock solutions. Where pH recordings were not made (see Fig. 2) the amount of acid to be added, especially in the presence of phosphate and citrate, was determined separately.RESULTS AND DISCUSSION Figure 1A shows the effect of acidifying the medium with H2SO4 on the light transmission of the mitochondrial suspension. There is close correspondence between osmotic swelling as measured by per cent transmission (% T) and osmotic theory (7, 11), and assuming that this holds over the neutral pH range (pH 6-8), addition of H2S04 initiates a slow osmotic swelling. Dropping the pH to 4.3 causes an abrupt "shrinkage" which slows after a few sec and takes several min to stabilize. The transition point between swelling and shrinkage was in the range of pH 5.8 to 6. Shrinkage was secured using several acids and it may represent a physical response of membrane or matrix proteins to high proton concentration rather than solute efflux driven by an acid gradient. Previous investigations, however, showed a loss of mitochondrial water at low pH (6). We limited the present investigation to the swelling...

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