To elucidate the contribution of phospholipase A₂ (PLA₂) activity of notexin to its ability to perturb membranes, comparative studies on the interaction of notexin and guanidinated notexin (Gu-notexin) with egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk sphingomyelin (EYSM) and EYPC/EYSM/cholesterol vesicles were conducted. EYSM notably reduced the membrane-damaging activity of notexin against EYPC vesicles, but had an insignificant influence on that of Gu-notexin. Unlike the effects noted with notexin, inactivation of PLA₂ activity by EDTA led to a reduction in the ability of Gu-notexin to induce EYPC/EYSM vesicle leakage and to increase Gu-notexin-induced membrane permeability of EYPC/EYSM/cholesterol vesicles. The geometrical arrangement of notexin and Gu-notexin in contact with either EYPC/EYSM vesicles or EYPC/EYSM/cholesterol vesicles differed. Moreover, global conformation of notexin and Gu-notexin differed in either Ca²⁺-bound or metal-free states. These results indicate that notexin and Gu-notexin could induce membrane permeability without the involvement of PLA₂ activity, and suggest that guanidination alters the membrane-bound mode of notexin on damaging phospholipid vesicles containing sphingomyelin and cholesterol.

The goal of the present study is to elucidate the mechanism of quercetin on modulating Naja naja atra phospholipase A₂ (PLA₂) activities. Sphingomyelin inhibited PLA₂ enzymatic activity and membrane-damaging activity against egg yolk phosphatidylcholine (EYPC), while cholesterol and quercetin abrogated the sphingomeyelin inhibitory effect. Quercetin incorporation led to a reduction in PLA₂ enzymatic activity and membrane-damaging activity toward EYPC/sphingomyelin/cholesterol vesicles. Both cholesterol and quercetin increased detergent resistance and reduced membrane fluidity of EYPC/sphingomyelin vesicles. Quercetin reduced detergent insolubility but increased ordered lipid packing of EYPC/sphingomyelin/cholesterol vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that quercetin altered the membrane-bound mode of PLA₂ differently upon absorption onto the membrane bilayers of different lipid compositions. However, 8-anilinonaphthalene sulphonate-binding assay revealed that quercetin marginally affected the interaction between active site of PLA₂ with phospholipid vesicles. Collectively, our data indicate that membrane-inserted quercetin modulates PLA₂ interfacial activity and membrane-damaging activity via its effects on membrane structure and membrane-bound mode of PLA₂.