Effect of Calcium on Kinetic and Structural Aspects of Dilution-Induced Micellar to Lamellar Phase Transformation in Phosphatidylcholine-Cholate Mixtures

Previously, we have shown [Almog, S., Kushnir, T., Nir, S., & Lichtenberg, D. (1986) Biochemistry 25, 2597–2605] that the distribution of cholate between phosphatidylcholine (PC) vesicles and aqueous media apparently obeys a single distribution coefficient, K. In PC-cholate mixed micellar systems, the monomer concentration does not rise much above the cholate's critical micelle concentration (cmc). Consequently, for vesicular systems, the cholate:PC molar ratio in the mixed aggregates (R_c) is given by R_e= [cholate]/([PC] + 1 /K) whereas for mixed micellar systems R_e= ([cholate] - cmc)/[PC]. Dilution of mixed micellar systems results in a decrease of R_e, due to an increase in the fraction of monomeric PC. If the decrease in R_eis to values lower than 0.3, micellar to lamellar transformation occurs. This process involves a sequence of three steps, namely, micellar equilibration followed by vesiculation and subsequent vesicle size growth via a lipid transfer mechanism. The ultimate size of the resultant vesicles is an increasing function of R_e. This work is devoted to the effect of calcium on the dilution-induced vesicle formation. Its major findings and conclusions are as follows: (i) Calcium reduces the cmc of the detergent and raises its distribution coefficient between PC vesicles and the aqueous medium. Thus, for any given cholate and PC concentrations, calcium causes an increase of R_e. (ii) The rate of all the steps which ultimately lead to an apparent equilibrium vesicle size distribution increases dramatically with increasing calcium concentration. Thus, equilibration is attained in seconds to minutes rather than many hours required in the absence of calcium, (iii) The rate of the postvesiculation size growth is an increasing function of lipid concentration, indicating the probable involvement of a fusion mechanism in the vesicle size equilibration. This conclusion is strengthened by retention of dextran, entrapped in unilamellar vesicles during cholate-induced size growth. (iv) Calcium has no effect on the dependence of vesicle size on R_e. Calcium, indeed, affects the aggregation state of the lipid by increasing the value of R_e, but for any given R_e, the state of aggregation is independent of calcium. This means that, for mixtures with low values of R_e, addition of calcium causes an increase of the mean diameter of the vesicles, whereas for higher R_evalues, it may lead to micellization and a subsequent decrease of micelle sizes. The lack of a direct effect of calcium on the state of aggregation of PC-cholate mixtures of any effective ratio, obtained in spite of the calcium-induced change in the mechanism of vesicle size growth, supports the conclusion of Schurtenberger et al. [Schurtenberger, P., Mazer, N. A., & Kanzig, W. (1985) J. Phys. Chem. 89, 1042–1049] that the ultimate size distribution obtained for PC-cholate vesicles represents a true thermodynamic equilibrium of the diluted systems.