Melittin-mediated release of [H-3]-oleic acid from E-coli cells is dependent upon heat- and trypsin-sensitive factor(s) in human serum

Synthetic melittin mediated the release of [H-3]-oleic acid ([H-3]-OA) or i ts acylated lipids from [H-3]-OA-labeled E. coli cells exposed to human ser um. This phenomenon was not observed in the absence of serum and was calciu m independent. The addition of serum was not required for melittin-mediated lysis of erythrocytes, although lysis was greater in the presence of serum than in its absence (P < 0.001). Trypsin treatment of human serum reduced the melittin-mediated release of [H-3]-OA/acylated lipids, and this effect was more pronounced upon boiling the serum (P < 0.01). A kinetic study show ed that maximum release of [H-3]-OA/acylated lipids occurred within 3-6 min . Thin layer chromatography (TLC) analysis showed the lipids to be phosphat idyl ethanolamine (PE), phosphatidylethanol (PEt) and phosphatidic acid (PA ). There was no detectable level of oleic acid (OA), diacylglycerol (DAG), phosphatidyl choline (PC) or phosphatidyl serine (PS). These findings sugge sted that a trypsin and heat-sensitive enzyme/factor present in the serum h ad a role in melittin-mediated action. These findings further showed that m elittin activated phospholipase D (PLD), without affecting phospholipase A( 2) (PLA(2)) or phospholipase C (PLC) activity. (C) 2000 Elsevier Science Lt d. All rights reserved.