Isohashi F, Nakanishi Y, Sakamoto Y
Biochemistry. 1983 Feb 1;22(3):584-90. doi: 10.1021/bi00272a010.
An acetyl-CoA hydrolase that is labile at low temperature was purified to homogeneity from the supernatant fraction of rat liver. The monomeric molecule, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had a molecular weight of about 63 000, while that of the purified enzyme, estimated by gel filtration, was 135 000. Thus, the enzyme consists of two subunits of identical molecular weight. On addition of adenosine 5'-triphosphate (ATP) or adenosine 5'-diphosphate (ADP) at 25 degrees C, the dimeric form of the enzyme aggregated to tetrameric forms (Mr 242 000 and Mr 230 000, respectively), whereas addition of adenosine 5'-monophosphate had little effect on enzyme association (Mr 145 000). When ATP was removed from the ATP-treated tetrameric enzyme by dialysis, the tetramer was mostly dissociated into the dimeric form. The apparent Km values for acetyl coenzyme A of the dimeric enzyme and tetrameric enzyme, reconstituted from the former in the presence of 2 mM ATP, were 170 microM and 60 microM, respectively. The purified dimeric enzyme was inactivated by exposure to lower temperature, especially below 10 degrees C. The various nucleotides tested partially stabilize the dimeric enzyme at low temperature, ATP being the most effective. Sucrose density gradient centrifugation showed that loss of catalytic activity by cold treatment was accompanied by dissociation of the dimer and tetramer into protomer.
一种在低温下不稳定的乙酰辅酶A水解酶从大鼠肝脏的上清液部分被纯化至同质。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计，单体分子的分子量约为63000，而通过凝胶过滤估计的纯化酶的分子量为135000。因此，该酶由两个分子量相同的亚基组成。在25℃下添加腺苷5'-三磷酸（ATP）或腺苷5'-二磷酸（ADP）时，酶的二聚体形式聚合成四聚体形式（分别为Mr 242000和Mr 230000），而添加腺苷5'-单磷酸对酶的缔合影响很小（Mr 145000）。当通过透析从ATP处理的四聚体酶中去除ATP时，四聚体大多解离成二聚体形式。由二聚体酶在2 mM ATP存在下重构得到的四聚体酶和二聚体酶对乙酰辅酶A的表观Km值分别为170 microM和60 microM。纯化的二聚体酶在暴露于较低温度，尤其是低于10℃时会失活。所测试的各种核苷酸在低温下部分稳定二聚体酶，其中ATP最有效。蔗糖密度梯度离心表明，冷处理导致的催化活性丧失伴随着二聚体和四聚体解离成原体。
