Chapman K D, Moore T S
Botany Department, Louisiana State University, Baton Rouge 70803.
Biochim Biophys Acta. 1994 Feb 10;1211(1):29-36. doi: 10.1016/0005-2760(94)90135-x.
We recently reported that a novel acyltransferase activity (fatty acid: diacylphosphatidylethanolamine N-acyltransferase) synthesizes N-acylphosphatidylethanolamine (NAPE), an unusual derivative of phosphatidylethanolamine (PE), in microsomes of cotyledons of cotton seedlings by direct acylation of PE with free fatty acids (Chapman and Moore (1993) Plant Physiol. 102(3), 761-769). Here we report the solubilization of this membrane-bound NAPE synthase activity from cottonseed microsomes and the separation of three active isozymes with distinctly different characteristics. NAPE synthase activity was solubilized from NaCl-washed microsomal membranes by 0.2 mM dodecylmaltoside (DDM) at a 2:1 (w:w) detergent/protein ratio (assessed by enzyme activity after centrifugation at 150,000 x gmax, 1 h). Two sequential preparative isoelectric focussing separations of DDM-solubilized microsomal membrane proteins resulted in recovery of three distinct peaks of NAPE synthase activity--one at pH 6.3, one at pH 7.2, and one at pH 8.4 (designated A, B and C). These isozymes were purified 1148-fold (A), 269-fold (B), and 729-fold (C) from homogenates of cotton cotyledons. A 28 kDa subunit was enriched in all three isozyme fractions. Each of the isozymes exhibited unique kinetic properties with respect to palmitic acid and dioleoyl-PE. Each of the solubilized isozymes exhibited positive cooperativity toward palmitic acid (consistent with previous studies of NAPE synthase activity in intact microsomes) but not toward dioleyl-PE. Collectively, these results indicate that the synthesis of NAPE in cotton cotyledons is complex and has a potential for being a highly regulated process. The isolation of active NAPE synthase isozymes will provide the foundation for future studies into the physiological role of NAPE synthase (and NAPE) and the regulation of NAPE metabolism in membranes of plant cells.
我们最近报道，一种新型酰基转移酶活性（脂肪酸：二酰基磷脂酰乙醇胺N-酰基转移酶）通过游离脂肪酸对磷脂酰乙醇胺（PE）进行直接酰化，在棉花幼苗子叶的微粒体中合成N-酰基磷脂酰乙醇胺（NAPE），这是磷脂酰乙醇胺（PE）的一种特殊衍生物（查普曼和摩尔（1993年）《植物生理学》102(3)，761 - 769）。在此，我们报道了从棉籽微粒体中溶解这种膜结合的NAPE合酶活性，并分离出三种具有明显不同特性的活性同工酶。NAPE合酶活性通过0.2 mM十二烷基麦芽糖苷（DDM）以2:1（w:w）的去污剂/蛋白质比例从用NaCl洗涤过的微粒体膜中溶解出来（通过在150,000 x gmax离心1小时后的酶活性评估）。对DDM溶解的微粒体膜蛋白进行两次连续的制备性等电聚焦分离，得到了三个不同的NAPE合酶活性峰——一个在pH 6.3，一个在pH 7.2，一个在pH 8.4（分别命名为A、B和C）。这些同工酶从棉花子叶匀浆中纯化了1148倍（A）、269倍（B）和729倍（C）。一个28 kDa的亚基在所有三个同工酶组分中都得到了富集。每种同工酶在棕榈酸和二油酰-PE方面都表现出独特的动力学特性。每种溶解的同工酶对棕榈酸表现出正协同性（与之前对完整微粒体中NAPE合酶活性的研究一致），但对二油酰-PE没有。总体而言，这些结果表明棉花子叶中NAPE的合成是复杂的，并且有可能是一个高度受调控的过程。活性NAPE合酶同工酶的分离将为未来研究NAPE合酶（和NAPE）的生理作用以及植物细胞膜中NAPE代谢的调控提供基础。
