Cai S J, McAndrew R S, Leonard B P, Chapman K D, Pidgeon C
Department of Medicinal Chemistry, School of Pharmacy, Purdue University, West Lafayette, IN 47907, USA.
J Chromatogr A. 1995 Apr 7;696(1):49-62. doi: 10.1016/0021-9673(94)01113-s.
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.
N-酰基磷脂酰乙醇胺合酶（NAPES）是一种膜结合酶，存在于棉花幼苗中，其浓度占总蛋白的≤0.02%。使用固定化人工膜（IAM）色谱法，通过单一色谱步骤将NAPES纯化至电泳纯。用于纯化NAPES的IAM柱是etherIAM.PEC10/C3，该表面含有单层固定化磷脂酰乙醇胺（PE）。由于PE是NAPES天然底物的类似物，etherIAM.PEC10/C3柱作为该酶的亲和柱发挥作用。将来自棉花的经去污剂增溶的微粒体蛋白加载到etherIAM.PEC10/C3柱上，并用含有0.2 mM二肉豆蔻酰磷脂酰乙醇胺（DMPE）和2 mM十二烷基麦芽糖苷的缓冲流动相洗脱。如果从流动相中去除DMPE，则很少有NAPES功能活性被洗脱。流动相DMPE也是NAPES的底物，流动相和IAM表面都含有NAPES底物。流动相DMPE可能既作为影响蛋白质洗脱的表面活性剂型亲和置换配体，又作为NAPES功能活性的稳定因子。半制备型etherIAM.PEC10/C3（6.5×1.0 cm）柱的上样量约为5 mg经去污剂增溶的微粒体蛋白总量，蛋白质回收率是定量的。NAPES的这一步IAM纯化在银染聚丙烯酰胺凝胶上产生单一条带，NAPES比活性增加了3940倍。纯化的NAPES蛋白的分子量为64000。125I标记的[12-(4-叠氮水杨酸基)氨基]十二烷酸是NAPES的光反应性脂肪酸底物，用于确认蛋白质纯度。
