Bond L W, Pynadath T I
Biochim Biophys Acta. 1976 Oct 21;450(1):8-20. doi: 10.1016/0005-2760(76)90294-0.
Purified outer membrane of beef liver mitochondria was found to elongate medium chain fatty acyl-CoA primer by the incorporation of [1-14C]acetyl-CoA. This enzymic activity, extracted by Triton X-100, was purified 8-fold by ammonium sulfate fractionation followed by chromatography on a Sephadex column. Purified inner membrane, when processed through an identical purification procedure, yielded a second enzyme system which incorporated [1-14C]acetyl-CoA into long chain fatty acids in the presence of medium chain fatty acyl-CoA primer. This enzyme preparation was about four times as active as the preparation from the outer membrane, and used NADH as the reductant for the synthesis. The molecular weights of the inner and the outer membrane enzyme systems, estimated by gel filtration as well as sucrose density gradient centrifugation, were approx. 57 000 and 126 000, respectively. The partially purified outer membrane enzyme system required NADH and a medium chain acyl-CoA primer for the incorporation of [1-14C]acetyl-CoA into long chain fatty acids. KNC stimulated the reaction. NADPH could substitute for NADH only to a limited extent. Malonyl-CoA was ineffective as a substrate in this reaction. The optimum pH of the reaction was 7.2-7.6 in 0.1 M potassium phosphate buffer. Dithiothreitol, beta-mercaptoethanol, N-ethylmaleimide and high concentrations of ATP and acyl-CoA primer inhibited the reaction. The specificity for the acyl-CoA primer in the reaction was very broad. All the primers tested, C8 to C16, incorporated acetyl-CoA significantly. However, maximum incorporation was observed with dodecanoyl-CoA. Decanoyl-CoA was the best primer for the enzyme system isolated from the inner membrane. About 42% of the radioactivity in the fatty acids synthesized by the outer membrane enzyme system, from myristoyl-CoA and [1-C14]acetyl-CoA, was in palmitic acid. Of the remaining activity, 41% was in stearic acid and about 38% in longer-chain acids. Hence, the elongation of the primer fatty acid by one C2 unit appeared to be the predominant process in this synthesis. In the elongation of myristoyl-C0A by the inner membrane enzyme system, palmitic acid which constituted nearly 78% of the fatty acids synthesized, was the primary product.
研究发现，牛肝线粒体的纯化外膜可通过掺入[1-¹⁴C]乙酰辅酶A来延长中链脂肪酰辅酶A引物。用 Triton X-100提取的这种酶活性，经硫酸铵分级分离，然后在葡聚糖凝胶柱上进行层析，纯化了8倍。纯化的内膜经过相同的纯化程序后，产生了第二个酶系统，该系统在中链脂肪酰辅酶A引物存在的情况下，将[1-¹⁴C]乙酰辅酶A掺入长链脂肪酸中。这种酶制剂的活性约为外膜制剂的四倍，并使用NADH作为合成的还原剂。通过凝胶过滤以及蔗糖密度梯度离心法估计，内膜和外膜酶系统的分子量分别约为57000和126000。部分纯化的外膜酶系统需要NADH和中链酰基辅酶A引物才能将[1-¹⁴C]乙酰辅酶A掺入长链脂肪酸中。KNC可刺激该反应。NADPH仅在有限程度上可替代NADH。丙二酰辅酶A在该反应中作为底物无效。在0.1M磷酸钾缓冲液中，反应的最适pH为7.2 - 7.6。二硫苏糖醇、β-巯基乙醇、N-乙基马来酰亚胺以及高浓度的ATP和酰基辅酶A引物可抑制该反应。该反应中对酰基辅酶A引物的特异性非常广泛。所有测试的引物，C8至C16，均能显著掺入乙酰辅酶A。然而，月桂酰辅酶A的掺入量最高。癸酰辅酶A是从内膜分离的酶系统的最佳引物。外膜酶系统由肉豆蔻酰辅酶A和[1-C¹⁴]乙酰辅酶A合成的脂肪酸中，约42%的放射性存在于棕榈酸中。其余活性中，41%存在于硬脂酸中，约38%存在于长链酸中。因此，引物脂肪酸延长一个C2单位似乎是该合成过程中的主要过程。在内膜酶系统使肉豆蔻酰辅酶A延长的过程中，占合成脂肪酸近78%的棕榈酸是主要产物。
