Calcium and Proton Transport in Membrane Vesicles from Barley Roots

Abstract

Ca2+ uptake by membrane fractions from barley (Hordeum vulgare L. cv CM72) roots was characterized. Uptake of 45Ca2+ was measured in membrane vesicles obtained from continuous and discontinuous sucrose gradients. A single, large peak of Ca2+ uptake coincided with the peak of proton transport by the tonoplast H+-ATPase. Depending on the concentration of Ca2+ in the assay, Ca2+ 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 Ca2+ uptake in the tonoplast-enriched fraction was not sensitive to ionophores but was inhibited by vanadate. The results suggest that 45Ca uptake was driven by the low affinity, high capacity tonoplast Ca2+/nH+ antiporter and also by a high affinity, lower capacity Ca2+-ATPase. The Ca2+-ATPase may be associated with tonoplast, Golgi or contaminating vesicles of unknown origin. No Ca2+ transport was specifically associated with the distinct peak of endoplasmic reticulum that was identified by NADH cytochrome c reductase, choline phosphotransferase, and dolichol-P-mannosyl synthase activities. A small shoulder of Ca2+ 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 Ca2+-ATPase. Vesicle volumes were estimated using electron spin resonance techniques, and intravesicular Ca2+ concentrations were estimated to be as high as 5 millimolar. ATP-driven uptake of Ca2+ created 800- to 2000-fold concentration gradients within minutes. Problems in interpreting the effects of Ca2+ on ATP-generated pH gradients are discussed and the suggestion is made that Ca2+ dissipates pH gradients by a different mechanism than is responsible for Ca2+ uptake into tonoplast vesicles.