McAndrew R S, Leonard B P, Chapman K D
University of North Texas, Department of Biological Sciences, Denton 76203-0218, USA.
Biochim Biophys Acta. 1995 Jun 6;1256(3):310-8. doi: 10.1016/0005-2760(95)00038-e.
N-Acylphosphatidylethanolamine (NAPE), an unusual acylated derivative of phosphatidylethanolamine (PE), is synthesized from free fatty acids and PE in cotton seedlings (Chapman and Moore (1993) Plant Physiol. 102(3), 761-769). Here we use a photoreactive dodecanoic acid analogue, [12-(4-azidosalicy)amino]dodecanoic acid (ASD), and its 125I-labeled derivative to identify a protein subunit which corresponds to this cottonseed NAPE synthase activity. Dodecylmaltoside (DDM)-solubilized microsomal NAPE synthase enzyme was irreversibly and progressively inactivated by adding increasing concentrations of ASD and illuminating with UV254 light. Protection from this photoinactivation was afforded by the natural substrate, palmitic acid. In low light, microsomal NAPE synthase utilized ASD as a substrate to synthesize NAPE; palmitic acid competed for this activity. NAPE synthase activity was measured directly in gel slices following nondenaturing PAGE of DDM-solubilized microsomal membrane proteins. Two-dimensional electrophoresis (nondenaturing PAGE, followed by SDS-PAGE) of photoaffinity-labeled, DDM-solubilized microsomal proteins revealed a 64 kDa polypeptide that was associated with the active NAPE synthase enzyme. Also, a 64 kDa protein was photoaffinity labeled in all NAPE synthase isozyme fractions isolated by preparative isoelectric focusing; photoaffinity labeling of this 64 kDa polypeptide was diminished in the presence of exogenously supplied palmitic acid. Collectively, our results demonstrate that ASD specifically interacts with NAPE synthase in a manner analogous to its fatty acid substrate and indicate that a 64 kDa polypeptide is a component of cottonseed microsomal NAPE synthase. ASD will be a useful molecular probe in future studies aimed at understanding the physiological role of this NAPE synthase enzyme in membranes of plant cells.
N-酰基磷脂酰乙醇胺（NAPE）是磷脂酰乙醇胺（PE）一种特殊的酰化衍生物，由棉苗中的游离脂肪酸和PE合成（查普曼和摩尔，《植物生理学》，1993年，第102卷第3期，761 - 769页）。在此，我们使用一种光反应性十二烷酸类似物，[12 -（4 - 叠氮水杨酸基）氨基]十二烷酸（ASD）及其125I标记衍生物，来鉴定一种与这种棉籽NAPE合酶活性相对应的蛋白质亚基。通过添加浓度递增的ASD并照射UV254光，十二烷基麦芽糖苷（DDM）增溶的微粒体NAPE合酶会被不可逆地逐步失活。天然底物棕榈酸可保护该酶免受这种光灭活作用。在弱光条件下，微粒体NAPE合酶利用ASD作为底物合成NAPE；棕榈酸可竞争此活性。在对DDM增溶的微粒体膜蛋白进行非变性聚丙烯酰胺凝胶电泳（PAGE）后，直接在凝胶切片中测定NAPE合酶活性。对光亲和标记的、DDM增溶的微粒体蛋白进行二维电泳（非变性PAGE，随后进行SDS - PAGE），结果显示一种64 kDa的多肽与活性NAPE合酶相关。此外，在通过制备性等电聚焦分离的所有NAPE合酶同工酶组分中，一种64 kDa的蛋白都被光亲和标记；在有外源供应的棕榈酸存在时，这种64 kDa多肽的光亲和标记减弱。总体而言，我们的结果表明ASD以类似于其脂肪酸底物的方式与NAPE合酶特异性相互作用，并表明一种64 kDa的多肽是棉籽微粒体NAPE合酶的一个组分。在未来旨在了解这种NAPE合酶在植物细胞膜中的生理作用的研究中，ASD将是一种有用的分子探针。
