Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Abstract

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 μm) and ATP(K _m (ATP)=3.9 μm), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl⁻ or NO ⁻₃ . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl⁻ was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ³⁻₄ which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl⁻-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl⁻-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl⁻-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.