title: Rapid purification of cotton seed membrane-bound N-acylphosphatidylethanolamine synthase by immobilized artificial membrane chromatography.
abstract: N-Acylphosphatidylethanolamine synthase (NAPES) is a membrane-bound enzyme present in cotton seedlings at a concentration of < or = 0.02% of the total protein. NAPES was purified to electrophoretic homogeneity in a single chromatographic step using immobilized artificial membrane (IAM) chromatography. The IAM column used for NAPES purification was etherIAM.PEC10/C3 and this surface contains a monolayer of immobilized phosphatidyl-ethanolamine (PE). Since PE is an analogue of the natural substrate for NAPES, etherIAM.PEC10/C3 columns function as an affinity column for this enzyme. Detergent-solubilized microsomal proteins from cotton were loaded on to the etherIAM.PEC10/C3 column and eluted with buffered mobile phases containing 0.2 mM dimyristoylphosphatidylethanolamine (DMPE) and 2 mM dodecylmaltoside. Little NAPES functional activity eluted if DMPE was removed from the mobile phase. Mobile phase DMPE is also a substrate for NAPES, both the mobile phase and IAM surface contains NAPES substrates. Mobile phase DMPE may function as both a surfactant-type affinity displacing ligand effecting protein elution and also a stabilizing factor of NAPES functional activity. The loading capacity on semi-preparative etherIAM.PEC10/C3 (6.5 x 1.0 cm) columns was ca. 5 mg of total detergent solubilized microsomal proteins, and protein recovery was quantitative. This one-step IAM purification of NAPES resulted in a single band on silver-stained polyacrylamide gels, and 3940 fold increase in NAPES specific activity. The molecular mass of the purified NAPES protein is 64,000. 125I labeled [12-(4-azidosalicyl)amino]dodecanoic acid is a photoreactive fatty acid substrate of NAPES that was used to confirm protein purity.
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Cai
authors.forename: R S
authors.initials: RS
authors.lastname: McAndrew
authors.forename: B P
authors.initials: BP
authors.lastname: Leonard
authors.forename: K D
authors.initials: KD
authors.lastname: Chapman
authors.forename: C
authors.initials: C
authors.lastname: Pidgeon
mesh_terms: D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002853:Chromatography, Liquid / Q000379:methods*; D003368:Gossypium / Q000201:enzymology*; D008567:Membranes, Artificial; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D008567:Membranes, Artificial; D000217:Acyltransferases; C042139:phosphatidylethanolamine N-acyltransferase