RAPID PURIFICATION OF COTTON SEED MEMBRANE-BOUND N-ACYLPHOSPHATIDYLETHANOLAMINE SYNTHASE BY IMMOBILIZED ARTIFICIAL MEMBRANE CHROMATOGRAPHY

N-Acylphosphatidylethanolamine synthase (NAPES) is a membrane-bound en zyme present in cotton seedlings at a concentration of less than or eq ual to 0.02% of the total protein. NAPES was purified to electrophoret ic homogeneity in a single chromatographic step using immobilized arti ficial membrane (IAM) chromatography. The IAM column used for NAPES pu rification was (ether)IAM.PE(C10/C3) and this surface contains a monol ayer of immobilized phosphatidylethanolamine (PE). Since PE is an anal ogue of the natural substrate for NAPES, (ether)IAM.PE(C10/C3) columns function as an affinity column for this enzyme. Detergent-solubilized microsomal proteins from cotton were loaded on to the (ether)IAM.PE(C 10/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, and the refore both the mobile phase and IAM surface contains NAPES substrates . Mobile phase DMPE may function as both a surfactant-type affinity di splacing ligand effecting protein elution and also a stabilizing facto r of NAPES functional activity. The loading capacity on semi-preparati ve (ether)IAM.PE(C10/C3) (6.5 X 1.0 Cm) columns was ca. 5 mg of total detergent solubilized microsomal proteins, and protein recovery was qu antitative. This one-step IAM purification of NAPES resulted in a sing le band on silver-stained polyacrylamide gels, and 3940 fold increase in NAPES specific activity. The molecular mass of the purified NAPES p rotein is 64000. I-125 labeled [12-(4-azidosalicyl)amino]dodecanoic ac id is a photoreactive fatty acid substrate of NAPES that was used to c onfirm protein purity.