Watkins P A, Howard A E, Gould S J, Avigan J, Mihalik S J
Kennedy Krieger Research Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
J Lipid Res. 1996 Nov;37(11):2288-95.
In Refsum disease, disorders of peroxisome biogenesis, and rhizomelic chondrodysplasia punctata, pathological accumulation of phytanic acid results from impaired alpha-oxidation of this branched-chain fatty acid. Previous studies from this laboratory indicated that activation of phytanic acid to its CoA derivative precedes its alpha-oxidation in peroxisomes. It was reported that this reaction is catalyzed by a unique phytanoyl-CoA synthetase in human peroxisomes. We wanted to determine whether phytanic acid activation in rats required long-chain acyl-CoA synthetase (LCS), very long-chain acyl-CoA synthetase (VLCS), or a different enzyme. To test directly whether LCS could activate phytanic acid, rat liver cDNA encoding this enzyme was transcribed and translated in vitro. The expressed enzyme had both LCS activity (assayed with palmitic acid, C16: 0) and phytanoyl-CoA synthetase activity; VLCS activity (assayed with lignoceric acid, C24: 0) was not detectable. The ratio of phytanoyl-CoA synthetized activity to palmitoyl-CoA synthetase activity for LCS synthetized in vitro (approximately 205) was higher than that observed in peroxisomes isolated from rat liver (5-10%), suggesting that the expressed enzyme contained sufficient phytanoyl-Coa synthetase activity to account for all activity observed in intact peroxisomes. Further experiments were carried out to verify that phytanic acid was activated by LCS in rat liver peroxisomes. Attempts to separate LCS from phytanoyl-CoA synthetase by chromatography on several matrices were unsuccessful. Preparative isoelectric focusing revealed that phytanoyl-CoA synthetase and LCS had indistinguishable isoelectric points. Phytanoyl-CoA synthetase activity was inhibited by unlabeled palmitic acid but not by lignoceric acid. Heat treatment inactivated both phytanoyl-CoA and palmitoyl-CoA synthetase activities at similar rates. 5,8,11,14-Eicosatetraynoic acid inhibited activation of phytanic acid and palmitic acid in a parallel dose-dependent manner, whereas activation of lignoceric acid was not affected. These data support our conclusion that rat liver LCS, an enzyme known to be present in peroxisomal membranes, has phytanoyl-CoA synthetase activity.
在雷夫叙姆病、过氧化物酶体生物发生障碍和点状软骨发育不良中，植烷酸的病理性蓄积是由于这种支链脂肪酸的α-氧化受损所致。本实验室先前的研究表明，植烷酸在过氧化物酶体中被激活生成其辅酶A衍生物后才会发生α-氧化。据报道，该反应由人过氧化物酶体中一种独特的植烷酰辅酶A合成酶催化。我们想确定大鼠体内的植烷酸激活是否需要长链酰基辅酶A合成酶（LCS）、极长链酰基辅酶A合成酶（VLCS）或其他不同的酶。为了直接测试LCS是否能激活植烷酸，编码该酶的大鼠肝脏cDNA在体外进行转录和翻译。表达的酶同时具有LCS活性（用棕榈酸，C16:0检测）和植烷酰辅酶A合成酶活性；未检测到VLCS活性（用二十四烷酸，C24:0检测）。体外合成的LCS的植烷酰辅酶A合成活性与棕榈酰辅酶A合成酶活性之比（约为205）高于从大鼠肝脏分离的过氧化物酶体中观察到的比值（5 - 10%），这表明表达的酶含有足够的植烷酰辅酶A合成酶活性，足以解释在完整过氧化物酶体中观察到的所有活性。进一步的实验旨在验证大鼠肝脏过氧化物酶体中的植烷酸是否由LCS激活。尝试通过在几种基质上进行色谱分离来将LCS与植烷酰辅酶A合成酶分开，但未成功。制备性等电聚焦显示，植烷酰辅酶A合成酶和LCS具有无法区分的等电点。植烷酰辅酶A合成酶活性受到未标记棕榈酸的抑制，但不受二十四烷酸的抑制。热处理以相似的速率使植烷酰辅酶A和棕榈酰辅酶A合成酶活性失活。5,8,11,14-二十碳四炔酸以平行的剂量依赖性方式抑制植烷酸和棕榈酸的激活，而二十四烷酸的激活不受影响。这些数据支持我们的结论，即大鼠肝脏LCS（一种已知存在于过氧化物酶体膜中的酶）具有植烷酰辅酶A合成酶活性。
