Ceramides modulate protein kinase C activity and perturb the structure of phosphatidylcholine/phosphatidylserine bilayers

We studied the effects of natural ceramide and a series of ceramide analogs with different acyl chain lengths on the activity of rat brain protein kin ase C (PKC) and on the structure of bovine liver phosphatidylcholine (BLPC) /dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylserine (DPPS) (3:1:1 molar ratio) bilayers using H-2-NMR and specific enzymatic assays i n the absence or presence of 7.5 mol % diolein (DO). Only a slight activati on of PKC was observed upon addition of the short-chain ceramide analogs (C -2-, C-6-, or C-8-ceramide); natural ceramide or C-16-ceramide had no effec t. In the presence of 7.5 mol % DO, natural ceramide and C-16-ceramide anal og slightly attenuated DO-enhanced PKC activity. H-2-NMR results demonstrat ed that natural ceramide and C-16-ceramide induced lateral phase separation of gel-like and liquid crystalline domains in the bilayers; however, this type of membrane perturbation has no direct effect on PKC activity. The add ition of both short-chain ceramide analogs and DO had a synergistic effect in activating PKC, with maximum activity observed with 20 mol % C-6-ceramid e and 15 mot % DO. Further increases in C-6-ceramide and/or DO concentratio ns led to decreased PKC activity. A detailed H-2-NMR investigation of the c ombined effects of C-6-ceramide and DO on lipid bilayer structure showed a synergistic effect of these two reagents to increase membrane tendency to a dopt nonbilayer structures, resulting in the actual presence of such struct ures in samples exceeding 20 mol % ceramide and 15 mol % DO. Thus, the incr eased tendency to form nonbilayer lipid phases correlates with increased PK C activity, whereas the actual presence of such phases reduced the activity of the enzyme. Moreover, the results show that short-chain ceramide analog s, widely used to study cellular effects of ceramide, have biological effec ts that are not exhibited by natural ceramide.