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

As revealed by resonance energy transfer utilizing pyrene-labeled phosphatidylcholine donor, the mainly electrostatically controlled binding of adriamycin (Adr) and cytochrome c (cyt c) to mixed egg yolk phosphatidic acid/phosphatidylcholine (eggPA/eggPC, 15:85 molar ratio) liposomes was reversed upon the inclusion of increasing contents of sphingosine. At a [sphingosine] / [eggPA] molar ratio of almost-equal-to 2:1, the degree of fluorescence quenching by cyt c and Adr was approximately the same as when using liposomes lacking eggPA. Similarly, the increase in the surface pressure of sphingosine/eggPA monolayers on an air/water interface due to the membrane penetration of either cyt c or Adr was progressively reduced by increasing the content of sphingosine in the monolayers. The above critical [sphingosine]/[acidic phospholipid] stoichiometry yielding dissociation of the positively charged ligands 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 sphingosine could be involved. One eggPA (having maximally two negative charges) appears to require two molecules of sphingosine whereas the maximally singly charged eggPG is neutralized by one sphingosine. For comparison we also studied the effects of sphingosine on the phospholipase A2 catalyzed hydrolysis of the pyrene-labeled acidic alkyl-acyl phospholipid analog 1-octacosanyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphatidylmethanol (C28-O-PHPM) and the corresponding phosphatidylcholine (C28-O-PHPC). In the presence of low Ca2+ concentrations (almost-equal-to 50 nM) limiting the rate of the enzymatic reaction, sphingosine gradually inhibited the hydrolysis of phosphatidylcholine, and at 1:6 sphingosine/C28-O-PHPC a nearly complete lack of hydrolysis was evident. In contrast, the presence of about equimolar sphingosine in C28-O-PHPM enhanced by approximately 2-fold the hydrolysis of this lipid. This activation was followed by an inhibition until at approximately 1.1:1 [sphingosine]/[C28-O-PHPM] very little activity was detected. However, sphingosine did not prevent the penetration of PLA2 into monolayers of the nonhydrolyzable dialkylphosphatidic acid. Therefore, unlike for Adr and cyt c, sphingosine does not prevent the membrane association of PLA2 while the expression of the catalytic activity of this enzyme is inhibited by sphingosine. The latter is likely to result from changes in substrate surface potential due to the presence of sphingosine, a cationic amphiphile.