Jiménez-Monreal A M, Aranda F J, Micol V, Sánchez-Piñera P, de Godos A, Gómez-Fernández J C
Departamento de Bioquímica y Biología Molecular "A", Facultad de Veterinaria, Universidad de Murcia, Spain.
Biochemistry. 1999 Jun 15;38(24):7747-54. doi: 10.1021/bi983062z.
The activation of protein kinase C alpha was studied by using a lipid system consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) (molar ratio 4:1) and different proportions of 1-palmitoyl-2-oleoyl-sn-glycerol (POG). The phase behavior of the lipidic system was characterized by using differential scanning calorimetry and 31P NMR, and a phase diagram was elaborated. The results suggested the formation of two diacylglycerol/phospholipid complexes, one at 15 mol % of POG and the second at 30 mol % of POG. These two complexes would define the three regions of the phase diagram: in the first region (concentrations of POG lower than 15 mol %) there is gel-gel immiscibility at temperatures below that of the phase transition between C1 and pure phospholipid, and a fluid lamellar phase above of the phase transition. In the second region (between 15 and 30 mol % of POG), gel-gel immiscibility between C1 and C2 with fluid-fluid immiscibility was observed, while inverted hexagonal HII and isotropic phases were detected by 31P NMR. In the third region (concentrations of POG higher than 30 mol %), gel-gel immiscibility seemed to occur between C2 and pure POG along with fluid-fluid immiscibility, while an isotropic phase was detected by 31P NMR. When PKC alpha activity was measured, as a function of POG concentration, maximum activity was found at POG concentrations as low as 5-10 mol %; the activity slightly decreased as POG concentration was increased to 45 mol % at 32 degrees C (above Tc) whereas activity did not change with increasing concentrations of POG at 5 degrees C (below Tc). When the activity was studied as a function of temperature, at different POG concentrations, and depicted as Arrhenius plots, it was found that the activity increased with increasing temperatures, showing a discontinuity at a temperature very close to the phase transition of the system and a lower activation energy at the upper slope of the graph, indicating that the physical state of the membrane affected the interaction of PKC alpha with the membrane.
通过使用由1-棕榈酰-2-油酰基-sn-甘油-3-磷酸胆碱（POPC）/1-棕榈酰-2-油酰基-sn-甘油-3-磷酸丝氨酸（POPS）（摩尔比4:1）和不同比例的1-棕榈酰-2-油酰基-sn-甘油（POG）组成的脂质体系，研究了蛋白激酶Cα的激活情况。通过差示扫描量热法和31P NMR对脂质体系的相行为进行了表征，并绘制了相图。结果表明形成了两种二酰基甘油/磷脂复合物，一种在POG含量为15 mol%时形成，另一种在POG含量为30 mol%时形成。这两种复合物定义了相图的三个区域：在第一个区域（POG浓度低于15 mol%），在低于C1与纯磷脂之间相变温度时存在凝胶-凝胶不混溶性，在相变温度以上为流体层状相。在第二个区域（POG含量在15%至30%之间），观察到C1和C2之间的凝胶-凝胶不混溶性以及流体-流体不混溶性，同时通过31P NMR检测到反相六角形HII相和各向同性相。在第三个区域（POG浓度高于30 mol%），C2与纯POG之间似乎发生凝胶-凝胶不混溶性以及流体-流体不混溶性，同时通过31P NMR检测到各向同性相。当测量PKCα活性作为POG浓度的函数时，在POG浓度低至5 - 10 mol%时发现最大活性；在32℃（高于Tc）时，随着POG浓度增加到45 mol%，活性略有下降，而在5℃（低于Tc）时，活性随POG浓度增加没有变化。当研究活性作为温度的函数，在不同POG浓度下，并绘制为阿仑尼乌斯图时，发现活性随温度升高而增加，在非常接近体系相变的温度处出现不连续性，并且在图的上斜率处具有较低的活化能，表明膜的物理状态影响PKCα与膜的相互作用。
