Respiration-Coupled Calcium Transport by Membrane Vesicles fromAzotobacter vinelandii

Abstract

Membrane vesicles, isolated from osmotic lysates of A. vinelandii spheroplasts in Tris-acetate buffer, rapidly accumulate Ca in the presence of an oxidizable substrate. The addition of D-lactate to vesicles increases the rate of Ca uptake 34-fold; L-malate, NADH, NADPH and reduced phenazine methosulfate are nearly as effective as lactate. The intravesicular Ca pool which accumulates under these conditions is rapidly discharged by isotopic exchange or in the presence of respiratory inhibitors, uncouplers or EGTA [ethyleneglycol-bis(aminoethylether)-N,N''-tetraacetic acid]. The uptake rates for Ca follow Michaelis-Menten kinetics yielding a Km of 48 .mu.M and a Vmax of 45 nmol/min per mg membrane protein. Initial rates of EGTA-induced Ca efflux also follow saturation kinetics, giving a Vmax identical to that for Ca entry; the Km for exodus is 14 mM, assuming that free Ca accumulates in vesicles. The difference in the affinity of Ca for the entry and exit processes observed during respiration is sufficient to account for the estimated 150-fold Ca concentration gradient achieved under steady-state conditions. The uptake system is specific for Ca as opposed to other cations, but Zn and La are effective competitors. Ca uptake is blocked when electron transport is inhibited by exposure of vesicles to p-chlormercuriphenylsulfonate, hydroxyquinoline-N-oxide or cyanide or under anoxic conditions. Divalent cation ionophores (A23187 and X537A) and proton ionophores (CCP [carbonyl cyanide-m-chlorophenylhydrazone] and gramicidin D) also block Ca transport effectively. The electrogenic K ionophore valinomycin has no effect on lactate-dependent Ca uptake in the presence of K; ionophores which induce electroneutral exchange of protons for Na or K (monensin and nigericin, respectively) block Ca transport in the presence of appropriate cation. The fluorescence intensity of quinacrine (an amine probe) in the presence of A. vinelandii membrane vesicles is reduced by 25% on addition of lactate; the quenching is blocked by CCP. this indicates that a pH gradient (inside acid) is developed across the vesicle membrane during lactate oxidation. These membrane preparations contain vesicles of inverted topology (with respect to the intact cell) and Ca transport occurs by electroneutral Ca/proton antiport.