Interaction of Ca 2+ with cardiolipin-containing liposomes and its inhibition by adriamycin

Abstract

The interaction of cardiolipin-containing, unilamellar liposomes with Ca 2+ was assessed by flow dialysis in the presence of 2–100 μM 45Ca 2+, using vesicles formed from phosphatidylcholine (PC) and from PC and cardiolipin in mole ratios from 16:1 to 1:1. Control (PC only) vesicles bound no detectable Ca 2+. In contrast, Ca 2+ binding to cardiolipin-containing vesicles was substantial and dependent on vesicle concentration. Scatchard plots for the binding were concave upward. Resolution of the data, assuming the presence of two independent classes of binding sites, indicated a high-affinity site with apparent k D = 5.57 ± 0.48 μM (S.D.) and a second site with K D in the millimolar range. Interaction of cardiolipin-containing liposomes with Ca 2+ was insensitive to monovalent cations (Na +, K +, Rb +), but was inhibited by ruthenium red ⪢ La 3+ > Mn 2+ > Mg 2+. Progressive increases in the PC: cardiolipin ratio markedly increased the apparent K D for Ca 2+ at the high-affinity site. Stoichiometry of Ca 2+ binding at the site passed through a maximum at a PC: cardiolipin ratio of 4:1. The potent antineoplastic agent adriamycin also inhibited the interaction of Ca 2+ with cardiolipin-containing liposomes in a dose-dependent manner; effects were detected at 10 μuM antibiotic. Unlike PC, adriamycin altered the Stoichiometry of the high-affinity interaction but not the apparent k D . Adriamycin effects increased with pH in the range of the p K A of its amino group. These results suggest that inhibition by adriamycin may result from a mechanism other than simple competition for the charged head group of cardiolipin.