Calcium and proton transport in membrane vesicles from barley roots

Ca(2+) uptake by membrane fractions from barley (Hordeum vulgare L. cv CM72) roots was characterized. Uptake of (45)Ca(2+) was measured in membrane vesicles obtained from continuous and discontinuous sucrose gradients. A single, large peak of Ca(2+) uptake coincided with the peak of proton transport by the tonoplast H(+)-ATPase. Depending on the concentration of Ca(2+) in the assay, Ca(2+) uptake was inhibited 50 to 75% by those combinations of ionophores and solutes that eliminated the pH gradient and membrane potential. However, 25 to 50% of the Ca(2+) uptake in the tonoplast-enriched fraction was not sensitive to ionophores but was inhibited by vanadate. The results suggest that (45)Ca uptake was driven by the low affinity, high capacity tonoplast Ca(2+)/nH(+) antiporter and also by a high affinity, lower capacity Ca(2+)-ATPase. The Ca(2+)-ATPase may be associated with tonoplast, Golgi or contaminating vesicles of unknown origin. No Ca(2+) transport was specifically associated with the distinct peak of endoplasmic reticulum that was identified by NADH cytochrome c reductase, choline phosphotransferase, and dolichol-P-man-nosyl synthase activities. A small shoulder of Ca(2+) uptake in the plasma membrane region of the gradient was inhibited by vanadate and erythrosin B and may represent the activity of a separate plasma membrane Ca(2+)-ATPase. Vesicle volumes were estimated using electron spin resonance techniques, and intravesicular Ca(2+) concentrations were estimated to be as high as 5 millimolar. ATP-driven uptake of Ca(2+) created 800- to 2000-fold concentration gradients within minutes. Problems in interpreting the effects of Ca(2+) on ATP-generated pH gradients are discussed and the suggestion is made that Ca(2+) dissipates pH gradients by a different mechanism than is responsible for Ca(2+) uptake into tonoplast vesicles.