ABSTRACT
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.
ABSTRACT
To elucidate the contribution of phospholipase A 2 (PLA 2 ) 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 insignifi cant infl uence on that of Gu-notexin. Unlike the effects noted with notexin, inactivation of PLA 2 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 PLA 2 activity, and suggest that guanidination alters the membrane-bound mode of notexin on damaging phospholipid vesicles containing sphingomyelin and cholesterol.
ABSTRACT
The aim of the present study is to explore whether membrane targeting of K+ channel-interacting protein 1 (KChIP1) is associated with its EF-hand motifs and varies with specifi c phospholipids. Truncated KChIP1, in which the EFhands 3 and 4 were deleted, retained the α-helix structure, indicating that the N-terminal half of KChIP1 could fold appropriately. Compared with wild-type KChIP1, truncated KChIP1 exhibited lower lipid-binding capability. Compared with wild-type KChIP1, increasing membrane permeability by the use of digitonin caused a marked loss of truncated KChIP1, suggesting that intact EF-hands 3 and 4 were crucial for the anchorage of KChIP1 on membrane. KChIP1 showed a higher binding capability with phosphatidylserine (PS) than truncated KChIP1. Unlike that of truncated KChIP1, the binding of wild-type KChIP1 with membrane was enhanced by increasing the PS content. Moreover, the binding of KChIP1 with phospholipid vesicles induced a change in the structure of KChIP1 in the presence of PS. Taken together, our data suggest that EF-hands 3 and 4 of KChIP1 are functionally involved in a specifi c association with PS on the membrane.