LIPID LATERAL HETEROGENEITY IN PHOSPHATIDYLCHOLINE PHOSPHATIDYLSERINE/DIACYLGLYCEROL VESICLES AND ITS INFLUENCE ON PROTEIN-KINASE-C ACTIVATION/

To test the hypothesis that the activation of protein kinase C (PKC) i s influenced by lateral heterogeneities of the components of the lipid bilayer, the thermotropic phase behavior of dimyristoylphosphatidylch oline (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO ) vesicles was compared with the activation of PKC by this system. Dif ferential scanning calorimetry (DSC) and Fourier transform infrared (F TIR) spectroscopy were used to monitor the main transition (i.e., the gel-to-fluid phase transition) as a function of mole fraction DO (chi( DO)) in DMPC/DO, DMPS/DO, and [DMPC/DMPS (1:1, mol/mol)]/DO multilamel lar vesicles (MLVs). In each case, when chi(DO) < similar to 0.3, DO s ignificantly broadened the main transition and shifted it to lower tem peratures; but when chi(DO) > similar to 0.3, the main transition beca me highly cooperative, i.e., narrow, again. The coexistence of overlap ping narrow and broad transitions was clearly evident in DSC thermogra ms from chi(DO) approximate to 0.1 to chi(DO) approximate to 0.3, with the more cooperative transition growing at the expense of the broader one as chi(DO) increased. FTIR spectroscopy, using analogs of DMPC an d DMPS with perdeuterated acyl chains, showed that the melting profile s of all three lipid components in [DMPC/DMPS (1:1, mol/mol)]/DO MLVs virtually overlay when chi(DO) = 0.33, suggesting that a new type of p hase, with a phospholipid/DO mole ratio near 2:1, is formed in this sy stem. Collectively, the results are consistent with the coexistence of DO-poor and DO-rich domains throughout the compositions chi(DO) appro ximate to 0.1 to chi(DO) approximate to 0.3, even at temperatures abov e the main transition. Comparison of the phase behavior of the binary mixtures with that of the ternary mixtures suggests that DMPS/DO inter actions may be more favorable than DMPC/DO interactions in the ternary system, especially in the gel state. PKC activity was measured using [DMPC/DMPS (1:1, mol/mol)]/DO MLVs as the lipid activator. At 35 degre es C (a temperature above the main transition of the lipids), PKC acti vity increased gradually with increasing chi(DO) from chi(DO) approxim ate to 0.1 to chi(DO) approximate to 0.4, and activity remained high a t higher DO contents. In contrast, at 2 degrees C (a temperature below the main transition), PKC activity exhibited a maximum between chi(DO ) approximate to 0.1 and chi(DO) approximate to 0.3, and at higher DO contents activity was essentially constant at 20-25% of the activity a t the maximum. We infer from these results that the formation of DO-ri ch domains is related to PKC activation, and when the lipid is in the gel state, the coexistence of DO-poor and DO-rich phases also contribu tes to PKC activation.