Bhushan A, Singh R P, Singh I
Arch Biochem Biophys. 1986 Apr;246(1):374-80. doi: 10.1016/0003-9861(86)90482-0.
Palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by microsomal membranes but lignoceric acid solubilized with Triton WR-1339 was not an effective substrate even though the detergent dispersed the same amount of these fatty acids and was also not inhibitory to the enzyme [I. Singh, R. P. Singh, A. Bhushan, and A. K. Singh (1985) Arch. Biochem. Biophys. 236, 418-426]. This observation suggested that palmitoyl-CoA and lignoceroyl-CoA may be synthesized by two different enzymes. We have solubilized the acyl-CoA ligase activities for palmitic and lignoceric acid of rat brain microsomal membranes with Triton X-100 and resolved them into three separate peaks (fractions) by hydroxylapatite chromatography. Fraction A (palmitoyl-CoA ligase) had high specific activity for palmitic acid and Fraction C (lignoceroyl-CoA ligase) for lignoceric acid. Specific activity of palmitoyl-CoA ligase for palmitic acid was six times higher than in Fraction C and specific activity of lignoceroyl-CoA ligase for lignoceric acid was four times higher than in Fraction A. At higher concentrations of Triton X-100 (0.5%), lignoceroyl-CoA ligase loses activity whereas palmitoyl-CoA ligase does not. Lignoceroyl-CoA ligase lost 60% of activity at 0.6% Triton X-100. Palmitoyl-CoA ligase (T1/2 of 4.5 min) is more stable at 40 degrees C than lignoceroyl-CoA ligase (T1/2 of 1.5 min). The pH optimum of palmitoyl-CoA ligase was 7.7 and that of lignoceroyl-CoA ligase was 8.4. Similar to our results with intact membranes, palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by palmitoyl-CoA ligase whereas lignoceric acid when solubilized with Triton WR-1339 was not able to act as substrate for lignoceroyl-CoA ligase. Since solubilized enzyme activities for synthesis of palmitoyl-CoA and lignoceroyl-CoA from microsomal membranes can be resolved into different fractions by column chromatography and demonstrate different properties, we suggest that in microsomal membranes palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes.
用曲拉通WR - 1339增溶的棕榈酸可被微粒体膜转化为棕榈酰辅酶A，但用曲拉通WR - 1339增溶的二十四烷酸即使该去污剂分散的这两种脂肪酸量相同且对该酶也无抑制作用，却不是有效的底物[I. 辛格、R. P. 辛格、A. 布尚和A. K. 辛格（1985年）《生物化学与生物物理学报》236卷，第418 - 426页]。这一观察结果表明，棕榈酰辅酶A和二十四烷酰辅酶A可能由两种不同的酶合成。我们用曲拉通X - 100增溶了大鼠脑微粒体膜中棕榈酸和二十四烷酸的酰基辅酶A连接酶活性，并通过羟基磷灰石层析将它们分离成三个单独的峰（组分）。组分A（棕榈酰辅酶A连接酶）对棕榈酸具有高比活性，组分C（二十四烷酰辅酶A连接酶）对二十四烷酸具有高比活性。棕榈酰辅酶A连接酶对棕榈酸的比活性比组分C高6倍，二十四烷酰辅酶A连接酶对二十四烷酸的比活性比组分A高4倍。在较高浓度的曲拉通X - 100（0.5%）下，二十四烷酰辅酶A连接酶失活而棕榈酰辅酶A连接酶不失活。在0.6%曲拉通X - 100时，二十四烷酰辅酶A连接酶丧失60%的活性。棕榈酰辅酶A连接酶（半衰期为4.5分钟）在40℃比二十四烷酰辅酶A连接酶（半衰期为1.5分钟）更稳定。棕榈酰辅酶A连接酶的最适pH为7.7，二十四烷酰辅酶A连接酶的最适pH为8.4。与我们对完整膜的研究结果相似，用曲拉通WR - 1339增溶的棕榈酸可被棕榈酰辅酶A连接酶转化为棕榈酰辅酶A，而用曲拉通WR - 1339增溶的二十四烷酸不能作为二十四烷酰辅酶A连接酶的底物。由于从微粒体膜中增溶的用于合成棕榈酰辅酶A和二十四烷酰辅酶A的酶活性可通过柱层析分离成不同组分并表现出不同特性，我们认为在微粒体膜中棕榈酰辅酶A和二十四烷酰辅酶A是由两种不同的酶合成的。
