A comparative study of the activation of protein kinase C alpha by different diacylglycerol isomers

The lipid activation of protein kinase C alpha (PKC alpha) has been studied by comparing the activation capacity of different 1,2-diacylglycerols and 1,3-diacylglycerols incorporated into mixed micelles or vesicles. Unsaturat ed 1,2-diacylglycerols were, in general, more potent activators than satura ted ones when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS)/Triton X-100 mixed micelles and pure POPS vesicles were used. In contrast, these differences were not observed when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosph ocholine (POPC)/POPS (4.1, molar ratio) vesicles were used. Diacylglycerols bearing short fatty acyl chains showed a very high activation capacity, ho wever, the capacity was less in mixed micelles. Furthermore, 1,2-diacylglyc erols had a considerably higher activating capacity than 1,3-diacylglycerol s in POPS/Triton X-100 mixed micelles and in POPC/POPS vesicles. However, t he differences between the two types of diacylglycerols were smaller when p ure POPS vesicles were used. Differential scanning calorimetry (DSC) showed that POPC/POPS membrane samples containing diacylglycerols had endothermic transitions in the presence of 200 mu M Ca2+ and 5 mM Mg2+. Transitions we re not detected when using pure POPS vesicles due to the formation of dehyd rated phases as demonstrated by FTIR (Fourier-transform infrared) spectrosc opy. PKC alpha binding studies, performed by differential centrifugation in the presence of 200 mu M Ca2+ and 5 mM Mg2+, showed that 1,2-sn-dioleoylgl ycerol (1,2-DOG) was more effective than 1,3-dioleoylglycerol (1,3-DOG) in promoting binding to POPC/POPS vesicles. However, when pure POPS vesicles w ere used, PKC alpha was able to bind to membranes containing either 1,2-DOG or 1,3-DOG to the same extent.