球形红假单胞菌光合膜的磷脂拓扑结构

Phospholipid topography of the photosynthetic membrane of Rhodopseudomonas sphaeroides.

作者信息

Al-Bayatti K K, Takemoto J Y

出版信息

Biochemistry. 1981 Sep 15;20(19):5489-95. doi: 10.1021/bi00522a022.

Abstract

The topography of phospholipids in the photosynthetic membranes of Rhodopseudomonas sphaeroides was investigated by using purified chromatophores and spheroplast-derived vesicles (SDVs). Chromatophores are closed vesicles oriented inside out with respect to the cytoplasmic membrane (cytoplasmic side out) and obtained from French-pressed cell lysates. SDVs are oriented right side out (periplasmic side out) and are obtained after osmotic lysis of lysozyme-treated cells. Phosphatidylethanolamine (PE) comprised approximately 62% and phosphatidylglycerol (PG) comprised approximately 33% of the total phospholipid of both vesicle preparations. The relatively membrane impermeable reagent trinitrobenzenesulfonate (TNBS) at 3 mM concentration and 5 degrees C modified chromatophore and SDV PE with kinetics indicating the occurrence of fast- and slow-reacting pools of PE. The fast-reacting pools comprised 33% and 55% of the total PE of chromatophores and SDVs, respectively. The slow-reacting pools comprised 61% and 32% of the total PE of chromatophores and SDVs, respectively. Phospholipase A2 treatment of chromatophores (1 unit/mg of vesicle protein) for 1 h at 37 degrees C resulted in hydrolysis of 73% and 77% of the total PG and PE, respectively. Similar enzyme treatment of SDVs resulted in 14% and 60% hydrolysis of the total PG and PE, respectively. Phospholipase A2 treatment inhibited 60% of the succinate dehydrogenase activity of chromatophores but only 8% of the activity of SDVs, indicating the membrane impermeability of phospholipase A2. Incubation of chromatophores for 10 min with 3 mM TNBS at 5 degrees C and then treatment with phospholipase A2 for 10 min and 1 h resulted in the hydrolysis of 10% and 61%, respectively, of unmodified PE. The results indicate asymmetric distributions of PE polar head groups (32-33% cytoplasmic side, 55-61% periplasmic side) and PG (73% cytoplasmic side, 14% periplasmic side) across the membrane. Also, a rapid and unidirectional transbilayer movement of PE polar head groups from the periplasmic to cytoplasmic surfaces of the membrane appears to occur during phospholipase A2 hydrolysis on the chromatophore surfaces.

摘要

利用纯化的载色体和原生质球衍生囊泡（SDV）研究了球形红假单胞菌光合膜中磷脂的拓扑结构。载色体是相对于细胞质膜（细胞质面朝外）内外翻转的封闭囊泡，由法式压榨细胞裂解物获得。SDV是外表面朝外（周质面朝外）的囊泡，通过溶菌酶处理的细胞渗透裂解后获得。两种囊泡制剂中，磷脂酰乙醇胺（PE）约占总磷脂的62%，磷脂酰甘油（PG）约占33%。浓度为3 mM的相对膜不透性试剂三硝基苯磺酸（TNBS）在5℃下修饰载色体和SDV中的PE，动力学表明存在快速反应池和慢速反应池的PE。快速反应池分别占载色体和SDV总PE的33%和55%。慢速反应池分别占载色体和SDV总PE的61%和32%。在37℃下用磷脂酶A2（1单位/毫克囊泡蛋白）处理载色体1小时，分别导致总PG和PE水解73%和77%。对SDV进行类似的酶处理分别导致总PG和PE水解14%和60%。磷脂酶A2处理抑制了载色体琥珀酸脱氢酶活性的60%，但仅抑制了SDV活性的8%，表明磷脂酶A2的膜不透性。在5℃下将载色体与3 mM TNBS孵育10分钟，然后用磷脂酶A2处理10分钟和1小时，分别导致未修饰PE水解10%和61%。结果表明PE极性头部基团（32 - 33%在细胞质面，55 - 61%在周质面）和PG（73%在细胞质面，14%在周质面）在膜上呈不对称分布。此外，在载色体表面进行磷脂酶A2水解过程中，PE极性头部基团似乎从膜的周质面到细胞质面发生了快速且单向的跨膜运动。