Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue

Abstract

Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using ⁴⁵Ca²⁺. Uptake of ⁴⁵Ca²⁺ by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of ⁴⁵Ca²⁺ uptake to ATP utilization via δμH⁺, no evidence for a secondary H⁺/Ca²⁺ antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca²⁺ and an imposed pH gradient could not drive ⁴⁵Ca²⁺ uptake. Optimal uptake of ⁴⁵Ca²⁺ occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N′-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of ⁴⁵Ca²⁺ uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K_m values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving ⁴⁵Ca²⁺ uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell.