Wang Jun, Okamoto Yasuo, Tsuboi Kazuhito, Ueda Natsuo
Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan.
Neuropharmacology. 2008 Jan;54(1):8-15. doi: 10.1016/j.neuropharm.2007.06.001. Epub 2007 Jun 22.
N-Acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a membrane-bound enzyme which releases the endocannabinoid anandamide and other bioactive N-acylethanolamines from their corresponding NAPEs in animal tissues. Our previous studies showed that NAPE-PLD solubilized from the membrane is remarkably stimulated by millimolar concentrations of Ca(2+) while the membrane-bound form is much less sensitive to Ca(2+). This finding suggested that certain membrane constituents diminished the stimulatory effect of Ca(2+). In the present studies, we examined the effects of membrane fractions from COS-7 cells and brain tissue on the purified recombinant rat NAPE-PLD, and found that heat-stable membrane component(s) dose-dependently activated NAPE-PLD up to 4.8-5.0 fold. In the presence of the membrane fractions, however, the stimulatory effect of Ca(2+) on the purified NAPE-PLD was considerably reduced. When it was examined if the membrane fractions can be replaced with various pure phospholipids, phosphatidylethanolamine activated NAPE-PLD up to 3.3 fold, which was followed by decrease in the stimulatory effects of Ca(2+) and several other divalent cations. These results suggest that membrane components including phosphatidylethanolamine keep the membrane-associated form of NAPE-PLD constitutively active.
N-酰基磷脂酰乙醇胺（NAPE）水解磷脂酶D（NAPE-PLD）是一种膜结合酶，可在动物组织中从相应的NAPE释放内源性大麻素花生四烯乙醇胺和其他生物活性N-酰基乙醇胺。我们之前的研究表明，从膜上溶解的NAPE-PLD受到毫摩尔浓度的Ca(2+)的显著刺激，而膜结合形式对Ca(2+)的敏感性要低得多。这一发现表明某些膜成分减弱了Ca(2+)的刺激作用。在本研究中，我们检测了COS-7细胞和脑组织的膜组分对纯化的重组大鼠NAPE-PLD的影响，发现热稳定的膜成分以剂量依赖的方式激活NAPE-PLD，激活倍数高达4.8 - 5.0倍。然而，在存在膜组分的情况下，Ca(2+)对纯化的NAPE-PLD的刺激作用显著降低。当检测膜组分是否可以被各种纯磷脂替代时，磷脂酰乙醇胺激活NAPE-PLD的倍数高达3.3倍，随后Ca(2+)和其他几种二价阳离子的刺激作用降低。这些结果表明，包括磷脂酰乙醇胺在内的膜成分使NAPE-PLD的膜结合形式保持组成性活性。
