Lehner R, Kuksis A
Banting and Best Department of Medical Research, C. H. Best Institute, University of Toronto, Ontario, Canada.
J Biol Chem. 1993 Nov 25;268(33):24726-33.
We have purified to apparent homogeneity an acyl-CoA hydrolase activity from rat intestinal villus cell microsomes by heparin and anion exchange and affinity chromatography. The purified 54-kDa acyl-CoA hydrolase along with several microsomal proteins form a covalent acyl-protein bond upon incubation with an activated fatty acid (acyl-CoA). The acyl moiety of the acylated acyl-CoA hydrolase is stable to denaturation and extraction with organic solvents, but is displaced by neutral hydroxylamine or mercaptoethanol, indicating a labile high energy (thio)ester linkage. The enzyme activity is inhibited by thiol-directed reagents and activated by the presence of dithiothreitol suggesting the presence of a cysteine residue(s) at or near the active site. Common serine-esterase inhibitors (NaF, phenylmethylsulfonyl fluoride) and activators (Mg2+, Ca2+) had no effect on the hydrolase activity. The enzyme hydrolyzed (transferred to water) 14-20 carbon acyl-CoA with similar efficiencies and did not utilize glycerophospholipids or mono- and diacylglycerols as potential acyl donors/acceptors. Phospholipids and mono- and diradylglycerols at concentrations below 100 microM or polyclonal antibodies raised against the purified hydrolase did not inhibit the enzyme activity. However, the acyl-CoA hydrolase activity could be immunoprecipitated from solubilized microsomes or purified enzyme preparations with corresponding decrease of the hydrolase activity in the supernatant of the immunoprecipitate. Immunoblotting studies show cross-reactivity with a protein of an identical molecular mass in other rat or human tissues. It is concluded that the microsomal acyl-CoA hydrolase deserves consideration as a candidate acyl-enzyme intermediate in glycerolipid synthesis when associated with appropriate acyltransferases.
我们通过肝素、阴离子交换和亲和色谱法从大鼠肠绒毛细胞微粒体中纯化出一种酰基辅酶A水解酶活性，使其达到表观均一性。纯化后的54 kDa酰基辅酶A水解酶与几种微粒体蛋白在与活化脂肪酸（酰基辅酶A）孵育时形成共价酰基蛋白键。酰化酰基辅酶A水解酶的酰基部分对变性和有机溶剂提取稳定，但可被中性羟胺或巯基乙醇取代，表明存在不稳定的高能（硫）酯键。该酶活性受巯基导向试剂抑制，在二硫苏糖醇存在下被激活，这表明活性位点或其附近存在半胱氨酸残基。常见的丝氨酸酯酶抑制剂（氟化钠、苯甲基磺酰氟）和激活剂（Mg2+、Ca2+）对水解酶活性无影响。该酶以相似效率水解（转移至水中）14 - 20个碳原子的酰基辅酶A，且不利用甘油磷脂或单酰甘油和二酰甘油作为潜在的酰基供体/受体。浓度低于100 microM的磷脂、单酰甘油和二酰甘油或针对纯化水解酶产生的多克隆抗体均不抑制该酶活性。然而，酰基辅酶A水解酶活性可从溶解的微粒体或纯化的酶制剂中免疫沉淀，免疫沉淀物上清液中的水解酶活性相应降低。免疫印迹研究表明，在其他大鼠或人类组织中与相同分子量的蛋白质有交叉反应。结论是，当与适当的酰基转移酶相关时，微粒体酰基辅酶A水解酶作为甘油脂质合成中酰基酶中间体的候选者值得考虑。
