Calculated by using the procedure described in [18]. The final concentration of

Calculated by using the procedure described in [18]. The final concentration of lipids in the reaction mixture was 625 mM. The reaction was started by the addition of 5 ml of PKCa (0.004 mg/ ml). After 30 min 1317923 at 25uC, the reaction was stopped with 1 ml of ice-cold 25 (w/v) trichloroacetic acid (TCA) and 1 ml of ice-cold 0.05 (w/v) bovine serum albumin. After precipitation on ice for 30 min, the protein precipitate was collected on a 2.5 cm glass filter (Sartorius, Gottingen, Germany) and washed with 10 ml of ?ice-cold 10 trichloroacetic acid. The amount of 32Pi incorporated in histone was measured by liquid scintillation counting. The linearity of the assay was confirmed from the time-course of histone phosphorylation over a 30 min period. Additional control experiments were run in the absence of calcium to measure basalExpression and Purification of Protein Kinase CaThe full length cDNA for rat PKCa was kindly provided by Profs. Ono and Nishizuka (Kobe, Japan). PKCa was cloned into the plasmid pFastBac HT (Invitrogen, Madrid, Spain). A 0.5 litre scale culture of Sf9 insect cells (Spodoptera frugiperda) at 2.16106 cells/ml was infected with the recombinant baculovirus.Cells were harvested 60 h postinfection (cell viability 80 ), pelleted at 4500 rpm for 20 min, and resuspended in buffer containing 25 mM Tris-HCl pH 7.5, 100 mM EGTA, 50 mM NaF, 100 mM 18204824 NaVO3, 1 Triton X-100, 10 glycerol, 150 mM NaCl, 1 mMPIP2 Activation of PKCakinase activity only adding EGTA without any CaCl2 with a reaction time of 30 minutes.Data AnalysisThe dependence of PKCa activity on the contents of the different activators in the model membranes was analyzed by a non-linear least squares fit to a modified Hill equation: y azVmax xn K0:5n zxnwhere y is the measured activity of PKCa, a is the activity in the absence of lipid or Ca2+ (background), Vmax is the lipid-stimulated activity, x is the concentration of the activator, K0.5 is the concentration of activator resulting in half maximal activity and n is the Hill coefficient. Standard errors for n, Vmax and K0.5, taken for three independent experiments, are reported.ResultsThe important contribution of PIP2 to PKCa enzymatic activity was clearly observed when it was Title Loaded From File studied as a function of Ca2+ concentration. A POPC/POPS molar ratio of about 4 was used in these assays since the concentration of POPS in the inner monolayer of eukaryotic plasma membranes, such as in erythrocyte or platelet cells, is roughly this [19?1]. The physiological concentration of PIP2 has been described to be around 1 mol of the total lipid of plasma membranes [22,23] and it is likely to be concentrated in the inner monolayer at 2 mol , which increase locally if it forms clusters or patches [24]. As regards diacylglycerol, the physiological levels of this lipid in Cucurbitacin I biomembranes were reviewed in [25]. For example, quantitative measurements of diacylglycerols present in stimulated cells have shown that they may reach 1.45 mol of the total lipid concentration [26] or about 2 mol [27]. So the concentrations of diacylglycerol used in this work can be considered physiological and well within the range of diacylglycerol concentrations used in standard procedures for PKC activation assays, which use values similar to those used here [28] or even as high as 11.5 mol with respect to total lipid [29] or as 19 mol [30] or 25 mol [31]. In enzymatic studies where the effect of lipid concentrations were studied, 200 mM Ca2+ was used in order t.Calculated by using the procedure described in [18]. The final concentration of lipids in the reaction mixture was 625 mM. The reaction was started by the addition of 5 ml of PKCa (0.004 mg/ ml). After 30 min 1317923 at 25uC, the reaction was stopped with 1 ml of ice-cold 25 (w/v) trichloroacetic acid (TCA) and 1 ml of ice-cold 0.05 (w/v) bovine serum albumin. After precipitation on ice for 30 min, the protein precipitate was collected on a 2.5 cm glass filter (Sartorius, Gottingen, Germany) and washed with 10 ml of ?ice-cold 10 trichloroacetic acid. The amount of 32Pi incorporated in histone was measured by liquid scintillation counting. The linearity of the assay was confirmed from the time-course of histone phosphorylation over a 30 min period. Additional control experiments were run in the absence of calcium to measure basalExpression and Purification of Protein Kinase CaThe full length cDNA for rat PKCa was kindly provided by Profs. Ono and Nishizuka (Kobe, Japan). PKCa was cloned into the plasmid pFastBac HT (Invitrogen, Madrid, Spain). A 0.5 litre scale culture of Sf9 insect cells (Spodoptera frugiperda) at 2.16106 cells/ml was infected with the recombinant baculovirus.Cells were harvested 60 h postinfection (cell viability 80 ), pelleted at 4500 rpm for 20 min, and resuspended in buffer containing 25 mM Tris-HCl pH 7.5, 100 mM EGTA, 50 mM NaF, 100 mM 18204824 NaVO3, 1 Triton X-100, 10 glycerol, 150 mM NaCl, 1 mMPIP2 Activation of PKCakinase activity only adding EGTA without any CaCl2 with a reaction time of 30 minutes.Data AnalysisThe dependence of PKCa activity on the contents of the different activators in the model membranes was analyzed by a non-linear least squares fit to a modified Hill equation: y azVmax xn K0:5n zxnwhere y is the measured activity of PKCa, a is the activity in the absence of lipid or Ca2+ (background), Vmax is the lipid-stimulated activity, x is the concentration of the activator, K0.5 is the concentration of activator resulting in half maximal activity and n is the Hill coefficient. Standard errors for n, Vmax and K0.5, taken for three independent experiments, are reported.ResultsThe important contribution of PIP2 to PKCa enzymatic activity was clearly observed when it was studied as a function of Ca2+ concentration. A POPC/POPS molar ratio of about 4 was used in these assays since the concentration of POPS in the inner monolayer of eukaryotic plasma membranes, such as in erythrocyte or platelet cells, is roughly this [19?1]. The physiological concentration of PIP2 has been described to be around 1 mol of the total lipid of plasma membranes [22,23] and it is likely to be concentrated in the inner monolayer at 2 mol , which increase locally if it forms clusters or patches [24]. As regards diacylglycerol, the physiological levels of this lipid in biomembranes were reviewed in [25]. For example, quantitative measurements of diacylglycerols present in stimulated cells have shown that they may reach 1.45 mol of the total lipid concentration [26] or about 2 mol [27]. So the concentrations of diacylglycerol used in this work can be considered physiological and well within the range of diacylglycerol concentrations used in standard procedures for PKC activation assays, which use values similar to those used here [28] or even as high as 11.5 mol with respect to total lipid [29] or as 19 mol [30] or 25 mol [31]. In enzymatic studies where the effect of lipid concentrations were studied, 200 mM Ca2+ was used in order t.