首页> 外文OA文献 >Catalytic Properties of a Newly Discovered Acyltransferase That Synthesizes N-Acylphosphatidylethanolamine in Cottonseed (Gossypium hirsutum L.) Microsomes.
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Catalytic Properties of a Newly Discovered Acyltransferase That Synthesizes N-Acylphosphatidylethanolamine in Cottonseed (Gossypium hirsutum L.) Microsomes.

机译:一种新发现的酰基转移酶的催化性质,该酰基转移酶在棉籽(棉)中微粒体中合成N-酰基磷脂酰乙醇胺。

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摘要

We recently demonstrated that cotyledons of cotton (Gossypium hirsutum L.) seedlings synthesize N-acylphosphatidylethanolamine (NAPE), an unusual acylated derivative of phosphatidylethanolamine (PE), during postgerminative growth (K.D. Chapman and T.S. Moore [1993] Arch Biochem Biophys 301: 21-33). Here, we report the discovery of an acyltransferase enzyme, fatty acid: diacylphosphatidylethanolamine N-acyltransferase (designated NAPE synthase), that synthesizes NAPE from PE and free fatty acids (FFA) in cottonseed microsomes. [14C]NAPE was synthesized from [14C]palmitic acid and endogenous PE in a time-, pH-, temperature-, and protein concentration-dependent manner. [14C]Palmitic acid was incorporated exclusively into the N-acyl position of NAPE. [14C]palmitoyl coenzyme A (CoA) and [14C]-dipalmitoyl phosphatidylcholine (PC) were poor acyl donors for the synthesis of NAPE (i.e. 200- and 3000-fold lower incorporation efficiency than palmitic acid, respectively). Synthesis of NAPE from palmitoyl-CoA and dipalmitoyl-PC was observed only after the release of FFA in microsomes. We observed a temperature optimum of 45[deg]C and a pH optimum of 8.0 for the synthesis of [14C]NAPE from [14C]palmitic acid (or from [14C]PE). NAPE synthase activity showed no apparent divalent cation requirement. Notably, activity was stimulated by HPO42-, HCO3-, SO42-, and NADPH, whereas activity was inhibited by Ca2+, Mn2+, Cd2+, ATP, ADP, flavin adenine disnucleotide, and flavin mononucleotide. Other nucleotide triphosphates (GTP and CTP) and pyridine dinucleotides (NAD, NADH, and NADP) did not appreciably affect NAPE synthase activity. Initial velocity measurements of NAPE synthase activity at increasing concentrations of palmitic acid showed non-Michaelis-Menten, biphasic kinetics. A high-affinity site (S0.5 = 7.2 [mu]M, Vmax = 18.8 nmol h-1 mg-1 of protein) and a low-affinity site (S0.5 = 32.0 [mu]M, Vmax = 44.9 nmol h-1 mg-1 of protein) were identified. Both sites exhibited positive cooperativity. Adding myristic, stearic, or oleic acids at equimolar amounts reduced the incorporation of [14C]palmitic acid into NAPE at low concentrations (10 [mu]M, high-affinity site) but not at high concentrations (50 [mu]M, low-affinity site), indicating that the two putative sites can be distinguished by their fatty acid preferences.
机译:最近,我们证明了棉花(棉(Gossypium hirsutum L.))的子叶在发芽后的生长过程中会合成N-酰基磷脂酰乙醇胺(NAPE),这是磷脂酰乙醇胺(PE)的一种不寻常的酰化衍生物(KD Chapman和TS Moore [1993] Arch Biochem Biophys 301:21)。 -33)。在这里,我们报告发现了一种酰基转移酶脂肪酸:二酰基磷脂酰乙醇胺N-酰基转移酶(指定为NAPE合酶),它可以从PE和棉籽微粒体中的游离脂肪酸(FFA)合成NAPE。 [14C] NAPE是由[14C]棕榈酸和内源性PE以时间,pH,温度和蛋白质浓度依赖性方式合成的。 [14C]棕榈酸仅掺入NAPE的N-酰基位置。 [14C]棕榈酰辅酶A(CoA)和[14C]-二棕榈酰磷脂酰胆碱(PC)是用于NAPE合成的弱酰基供体(分别比棕榈酸的掺入效率低200倍和3000倍)。仅在微粒体中释放FFA之后,才观察到由棕榈酰-CoA和二棕榈酰-PC合成NAPE的过程。我们观察到由[14C]棕榈酸(或由[14C] PE)合成[14C] NAPE的最适温度为45℃,最适pH为8.0。 NAPE合酶活性没有明显的二价阳离子需求。值得注意的是,HPO42-,HCO3-,SO42-和NADPH刺激了活性,而Ca2 +,Mn2 +,Cd2 +,ATP,ADP,黄素腺嘌呤二核苷酸和黄素单核苷酸则抑制了活性。其他三磷酸核苷酸(GTP和CTP)和吡啶二核苷酸(NAD,NADH和NADP)不会明显影响NAPE合酶的活性。在棕榈酸浓度增加的情况下,NAPE合酶活性的初始速度测量结果显示非Michaelis-Menten双相动力学。高亲和力位点(S0.5 = 7.2μM,Vmax = 18.8 nmol h-1 mg-1蛋白)和低亲和力位点(S0.5 = 32.0μM,Vmax = 44.9 nmol鉴定出h-1 mg-1蛋白)。两个站点都表现出积极的合作性。以等摩尔量添加肉豆蔻酸,硬脂酸或油酸可降低[14C]棕榈酸在低浓度(10μM,高亲和力位点)进入NAPE的结合,但在高浓度(50μM,低亲和力)时则不会-亲和位点),表示两个推定位点可以通过其脂肪酸偏好加以区分。

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