Membrane phospholipid synthesis in Escherichia coli. Purification, reconstitution, and characterization of sn-glycerol-3-phosphate acyltransferase.
Membrane phospholipid synthesis in Escherichia coli. Purification, reconstitution, and characterization of sn-glycerol-3-phosphate acyltransferase.
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DOI：
10.1016/s0021-9258(19)68570-4
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发表时间：
1981-11
期刊：
影响因子：
--
通讯作者：
P. R. Green;A. Merrill;R. Bell
中科院分区：
文献类型：
--
作者：
P. R. Green;A. Merrill;R. Bell
大肠杆菌的膜结合的sn-甘油-3-磷酸酰基转移酶从来自菌株vL 3/pvL 1的膜的Triton X-100提取物中纯化至接近同质（Lightner，V.A.，Larson，T. J.，Taillehman，P.，Kantor，G. D、Raetz，CR H.，贝尔河，巴西-地M.，和Modrich，P.（1980）d Biol. Chem. 255，9413-9420）。该菌株含有携带pZsB基因（酶的结构基因）的杂合质粒，并过量产生超过10倍的sn-甘油-3-磷酸酰基转移酶活性。在含有Triton X-100的缓冲液中进行纯化的每个阶段，用磷脂重建酶活性是必要的。在Matrex凝胶红A、辛基-琼脂糖CL-4 B和羟基磷灰石上进行色谱分析，在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳，得到含有83，000表观分子量的单一条带的制剂。最终可重构比活性超过6 pmol/min/mg。棕榈酰-CoA、油酰-CoA、棕榈酰-酰基载体蛋白（ACP）和顺式-异戊烯酰-ACP硫酯是底物。sn-甘油3-磷酸的表观Kc 1与棕榈酰-CoA或棕榈酰-ACP均为150 μ 1。与含不饱和脂肪酸的硫酯相比，含饱和脂肪酸的硫酯观察到更高的V，，值和更低的表观K，值。溶血磷脂酸是产生的唯一产物。对重组酶的测定条件进行了优化。牛血清白蛋白和额外的磷脂似乎减轻了棕榈酰辅酶A的抑制作用。重组E.大肠杆菌磷脂在0 ℃下10分钟内完成。活性取决于所用磷脂的量和类型。观察到对磷脂酰甘油和心磷脂的一些偏好。sn-甘油-3-磷酸酰基转移酶是一种完整的细胞质膜蛋白，含有约56%的疏水性氨基酸残基。1.15）的大肠杆菌催化膜磷脂形成的初始承诺步骤。这种酶活性的调节似乎可能控制存在的磷脂的量（1-7）并调节磷脂的浓度。
The membrane-bound sn-glycerol-3-phosphate acyltransferase of Escherichia coli was purified to near homogeneity from Triton X-100 extracts of membranes from strain vL3/pvL1 (Lightner, V. A., Larson, T. J., Tailleur, P., Kantor, G. D., Raetz, CR H., Bell, R. M., and Modrich, P.(1980) d Biol. Chem. 255, 9413-9420). This strain contains a hybrid plasmid bearing the pZsB gene, a structural gene of the enzyme, and overproduces sn-glycerol-3-phosphate acyltransferase activity more than 10-fold. At each stage of the purification performed in buffers containing Triton X-100, reconstitution of enzyme activity with phospholipids was necessary. Chromatography on Matrex Gel Red A, Octyl-Sepharose CL-4B, and hydroxylapatite yielded preparations containing a single band of 83,000 apparent molecular weight upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Final reconstitutable specific activities in excess of 6 pmol/min/mg were achieved. Palmitoyl-CoA, oleoyl-CoA, palmitoyl-acyl carrier protein (ACP), and cis-vaccenoyl-ACP thioesters were substrates. The apparent K,,, for sn-glycerol 3-phosphate was 150 p~ with either palmitoyl-CoA or palmitoyl-ACP. Higher V,, values and lower apparent K,,, values were observed for thioesters containing saturated fatty acids than were observed for thioesters containing unsaturated fatty acids. Lysophosphatidic acid was the only product produced. Assay conditions for the reconstituted enzyme were optimized. Bovine serum albumin and additional phospholipids appeared to lessen inhibition by palmitoyl-CoA. Reconstitution with E. coli phospholipids was complete in 10 min at 0 C. Activity depended on the amount and type of phospholipids employed. Some preference for phosphatidylglycerol and cardiolipin was observed. The sn-glycerol-3-phosphate acyltransferase, an integral cytoplasmic membrane protein, contained about 56% hydrophobic amino acid residues.The membrane-bound sa-glycerol-3-phosphate acyltransferase (EC 2.3. 1.15) of Escherichia coli catalyzes the initial committed step in the formation of membrane phospholipids. Regulation of this enzyme activity appears likely to control the amount of phospholipid present (1-7) and to regulate the