EFFECTS OF SPHINGOSINE ON PERIPHERAL MEMBRANE INTERACTIONS - COMPARISON OF ADRIAMYCIN, CYTOCHROME-C, AND PHOSPHOLIPASE-A2

As revealed by resonance energy transfer utilizing pyrene-labeled phos phatidylcholine donor, the mainly electrostatically controlled binding of adriamycin (Adr) and cytochrome c (cyt c) to mixed egg yolk phosph atidic acid/phosphatidylcholine (eggPA/eggPC, 15:85 molar ratio) lipos omes was reversed upon the inclusion of increasing contents of sphingo sine. At a [sphingosine] / [eggPA] molar ratio of almost-equal-to 2:1, the degree of fluorescence quenching by cyt c and Adr was approximate ly the same as when using liposomes lacking eggPA. Similarly, the incr ease in the surface pressure of sphingosine/eggPA monolayers on an air /water interface due to the membrane penetration of either cyt c or Ad r was progressively reduced by increasing the content of sphingosine i n the monolayers. The above critical [sphingosine]/[acidic phospholipi d] stoichiometry yielding dissociation of the positively charged ligan ds Adr or cyt c from membrane acidic phospholipids was shifted from 2: 1 to 1:1 upon substituting egg phosphatidylglycerol (eggPG) for eggPA. Accordingly, charge neutralization of the acidic phospholipids by sph ingosine could be involved. One eggPA (having maximally two negative c harges) appears to require two molecules of sphingosine whereas the ma ximally singly charged eggPG is neutralized by one sphingosine. For co mparison we also studied the effects of sphingosine on the phospholipa se A2 catalyzed hydrolysis of the pyrene-labeled acidic alkyl-acyl pho spholipid analog n-1-yl)]hexanoyl-sn-glycero-3-phosphatidylmethanol (C 28-O-PHPM) and the corresponding phosphatidylcholine (C28-O-PHPC). In the presence of low Ca2+ concentrations (almost-equal-to 50 nM) limiti ng the rate of the enzymatic reaction, sphingosine gradually inhibited the hydrolysis of phosphatidylcholine, and at 1:6 sphingosine/C28-O-P HPC a nearly complete lack of hydrolysis was evident. In contrast, the presence of about equimolar sphingosine in C28-O-PHPM enhanced by app roximately 2-fold the hydrolysis of this lipid. This activation was fo llowed by an inhibition until at approximately 1.1:1 [sphingosine]/[C2 8-O-PHPM] very little activity was detected. However, sphingosine did not prevent the penetration of PLA2 into monolayers of the nonhydrolyz able dialkylphosphatidic acid. Therefore, unlike for Adr and cyt c, sp hingosine does not prevent the membrane association of PLA2 while the expression of the catalytic activity of this enzyme is inhibited by sp hingosine. The latter is likely to result from changes in substrate su rface potential due to the presence of sphingosine, a cationic amphiph ile.