Isozymes of cottonseed microsomal N-acylphosphatidylethanolamine synthase: detergent solubilization and...

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.