大鼠肝脏乙酰乙酰辅酶A合成酶的纯化与特性分析
Purification and characterization of acetoacetyl-CoA synthetase from rat liver.
作者信息
Ito M, Fukui T, Kamokari M, Saito T, Tomita K
出版信息
Biochim Biophys Acta. 1984 Jul 6;794(2):183-93.
Acetoacetyl-CoA synthetase (acetoacetate:CoA ligase) was purified to electrophoretic homogeneity from a rat liver supernatant by ammonium sulfate fractionation and successive chromatographies on DEAE-Sepharose, Blue-Sepharose, Red-Sepharose, CoA-Sepharose, Ultrogel AcA-44, and DEAE-Sepharose once again. The purified enzyme had a specific activity of 2.3 mumol acetoacetyl-CoA formed per min per mg protein, which constituted a 960-fold purification compared to the crude extract, with a 7.4% yield. The enzyme absolutely required ATP, CoA, a monovalent cation (K+, Rb+, Cs+ or NH4+) and a divalent cation (Mg2+, Mn2+ or Ni2+) for its activity, yielding acetoacetyl-CoA, AMP and PPi in equimolar amounts. The molecular weight of the enzyme was approximately 80 000 as determined by Ultrogel AcA-34 gel filtration, and 71 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was active only on acetoacetate and to a lesser extent on L-(+)-3-hydroxybutyrate, and the Km values for acetoacetate, L-(+)-3-hydroxybutyrate, ATP and CoA were 8 microM, 75 microM, 60 microM and 10 microM, respectively.
通过硫酸铵分级分离以及先后在DEAE-琼脂糖、蓝色琼脂糖、红色琼脂糖、辅酶A-琼脂糖、超凝胶AcA-44和DEAE-琼脂糖上进行层析，从大鼠肝脏上清液中纯化出乙酰乙酰辅酶A合成酶（乙酰乙酸：辅酶A连接酶），使其达到电泳纯。纯化后的酶比活性为每分钟每毫克蛋白质形成2.3微摩尔乙酰乙酰辅酶A，与粗提物相比纯化了960倍，产率为7.4%。该酶的活性绝对需要ATP、辅酶A、一价阳离子（K⁺、Rb⁺、Cs⁺或NH₄⁺）和二价阳离子（Mg²⁺、Mn²⁺或Ni²⁺），反应生成等摩尔量的乙酰乙酰辅酶A、AMP和焦磷酸。通过超凝胶AcA-34凝胶过滤测定，该酶的分子量约为80000，在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳测定其分子量为71000。该酶仅对乙酰乙酸有活性，对L-(+)-3-羟基丁酸的活性较低，乙酰乙酸、L-(+)-3-羟基丁酸、ATP和辅酶A的米氏常数分别为8微摩尔、75微摩尔、60微摩尔和10微摩尔。
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