Huang H W, Goldberg E M, Zidovetzki R
Department of Biology, University of California, Riverside, California 92521 USA.
Biophys J. 1999 Sep;77(3):1489-97. doi: 10.1016/S0006-3495(99)76996-1.
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 kinase C (PKC) and on the structure of bovine liver phosphatidylcholine (BLPC)/dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylserine (DPPS) (3:1:1 molar ratio) bilayers using (2)H-NMR and specific enzymatic assays in the absence or presence of 7.5 mol % diolein (DO). Only a slight activation 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 effect. In the presence of 7.5 mol % DO, natural ceramide and C(16)-ceramide analog slightly attenuated DO-enhanced PKC activity. (2)H-NMR results demonstrated 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 addition of both short-chain ceramide analogs and DO had a synergistic effect in activating PKC, with maximum activity observed with 20 mol % C(6)-ceramide and 15 mol % DO. Further increases in C(6)-ceramide and/or DO concentrations led to decreased PKC activity. A detailed (2)H-NMR investigation of the combined effects of C(6)-ceramide and DO on lipid bilayer structure showed a synergistic effect of these two reagents to increase membrane tendency to adopt nonbilayer structures, resulting in the actual presence of such structures in samples exceeding 20 mol % ceramide and 15 mol % DO. Thus, the increased tendency to form nonbilayer lipid phases correlates with increased PKC activity, whereas the actual presence of such phases reduced the activity of the enzyme. Moreover, the results show that short-chain ceramide analogs, widely used to study cellular effects of ceramide, have biological effects that are not exhibited by natural ceramide.
我们使用（2）H-NMR和特定的酶促测定法，在不存在或存在7.5摩尔％二油精（DO）的情况下，研究了天然神经酰胺和一系列具有不同酰基链长度的神经酰胺类似物对大鼠脑蛋白激酶C（PKC）活性以及牛肝磷脂酰胆碱（BLPC）/二棕榈酰磷脂酰胆碱（DPPC）/二棕榈酰磷脂酰丝氨酸（DPPS）（摩尔比3:1:1）双层结构的影响。添加短链神经酰胺类似物（C(2)-、C(6)-或C(8)-神经酰胺）时，仅观察到PKC有轻微激活；天然神经酰胺或C(16)-神经酰胺无影响。在存在7.5摩尔％DO的情况下，天然神经酰胺和C(16)-神经酰胺类似物略微减弱了DO增强的PKC活性。（2）H-NMR结果表明，天然神经酰胺和C(16)-神经酰胺在双层中诱导了凝胶状和液晶域的横向相分离；然而，这种类型的膜扰动对PKC活性没有直接影响。短链神经酰胺类似物和DO的添加在激活PKC方面具有协同作用，在20摩尔％C(6)-神经酰胺和15摩尔％DO时观察到最大活性。C(6)-神经酰胺和/或DO浓度的进一步增加导致PKC活性降低。对C(6)-神经酰胺和DO对脂质双层结构的联合作用进行的详细（2）H-NMR研究表明，这两种试剂具有协同作用，可增加膜采用非双层结构的倾向，导致在超过20摩尔％神经酰胺和15摩尔％DO的样品中实际存在此类结构。因此，形成非双层脂质相的增加趋势与PKC活性增加相关，而此类相的实际存在则降低了酶的活性。此外，结果表明，广泛用于研究神经酰胺细胞效应的短链神经酰胺类似物具有天然神经酰胺未表现出的生物学效应。
