Schepers L, Casteels M, Verheyden K, Parmentier G, Asselberghs S, Eyssen H J, Mannaerts G P
Afdeling Farmacologie, Katholieke Universiteit Leuven, Belgium.
Biochem J. 1989 Jan 1;257(1):221-9. doi: 10.1042/bj2570221.
The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase. Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum. A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria. Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase. As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum. Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied. Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively. Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa. The pH curves of the synthetases did not coincide. Triton X-100 affected the activity of each of the synthetases differently. Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase. The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl. The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound. Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa. The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes.
研究了大鼠肝脏中三羟基粪甾烷酰辅酶A合成酶的亚细胞分布和特性，并将其与棕榈酰辅酶A合成酶和胆酰辅酶A合成酶的亚细胞分布和特性进行了比较。三羟基粪甾烷酰辅酶A合成酶和胆酰辅酶A合成酶几乎完全定位于内质网。三羟基粪甾烷酰辅酶A合成酶中数量上微不足道的一部分可能存在于线粒体中。将三羟基粪甾烷酰辅酶A转化为胆酰辅酶A的过氧化物酶体中不含三羟基粪甾烷酰辅酶A合成酶。如已知的那样，棕榈酰辅酶A合成酶分布在线粒体、过氧化物酶体和内质网中。还研究了三种合成活性对底物和辅因子（ATP、CoASH）的依赖性。胆酸和三羟基粪甾烷酸不抑制棕榈酰辅酶A合成酶；棕榈酸非竞争性抑制其他合成酶。同样，胆酸非竞争性抑制三羟基粪甾烷酸的活化，反之亦然。合成酶的pH曲线不一致。Triton X-100对每种合成酶活性的影响不同。三羟基粪甾烷酰辅酶A合成酶对焦磷酸抑制的敏感性低于胆酰辅酶A合成酶。用1 M NaCl处理不能使合成酶从微粒体膜中溶解，但可用Triton X-100或Triton X-100加NaCl使其溶解。通过在结合了三羟基粪甾烷酸的琼脂糖上进行亲和层析，可将去污剂溶解的三羟基粪甾烷酰辅酶A合成酶与溶解的胆酰辅酶A合成酶和棕榈酰辅酶A合成酶分离。在肾脏或肠黏膜的匀浆中未检测到胆酰辅酶A合成酶和三羟基粪甾烷酰辅酶A合成酶。结果表明，长链脂肪酸、胆酸和三羟基粪甾烷酸由三种不同的酶激活。
