Rat liver phosphofructokinase isozymes.
Rat liver phosphofructokinase isozymes.
复制标题
大鼠肝脏磷酸果糖激酶同工酶。
DOI：
10.1016/0003-9861(74)90224-0
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发表时间：
1974
影响因子：
3.9
通讯作者：
G. Weber
中科院分区：
文献类型：
--
作者：
G. Dunaway;G. Weber
发现大鼠肝脏不稳定的磷酸果糖激酶活性在 50 mmTris-HCl、pH 8.0、50 mmNaF、10 mm二硫苏糖醇和 1.0 mmATP 中得到稳定和有效提取。采用DEAE-纤维素层析法可以将肝磷酸果糖激酶活性拆分成两种同工酶。占总分离活性 85% 的主要同工酶被纯化至同质。这种纯化了 15,000 倍的同工酶的比活性约为 90 IU/mg 蛋白质，总活性回收率为 25-30%。十二烷基硫酸钠处理的同工酶的十二烷基硫酸钠-聚丙烯酰胺圆盘凝胶电泳显示亚基分子量为65,000。从家兔中获得主要同工酶的抗血清，两种同工酶的免疫滴定表明它们在免疫学上存在差异。两种同工酶的动力学特性表明，主要同工酶对 ATP 和柠檬酸盐抑制更敏感，并且通过果糖-6-P、AMP 和氨减轻 ATP 和柠檬酸盐抑制。通过DEAE-纤维素层析和100,000g上清液的抗血清滴定，表明这两种肝同工酶在成人、胚胎、肿瘤性肝和肾中均同时存在。
The labile phosphofructokinase activity of rat liver was found to be stabilized and efficiently extracted in 50 mmTris-HCl, pH 8.0, 50 mmNaF, 10 mmdithiothreitol, and 1.0 mmATP. By the method of DEAE-cellulose chromatography liver phosphofructokinase activity could be resolved into two isozymes. The major isozyme which was 85% of the total isolated activity was purified to homogeneity. This 15,000-fold purified isozyme had a specific activity of about 90 IU/mg protein with 25–30% recovery of the total activity. Sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of the sodium dodecyl sulfate-treated isozyme indicated a subunit molecular weight of 65,000. Antiserum to the major isozyme was obtained from rabbits, and immunotitration of the two isozymes indicated that they were immunologically different. Kinetic properties of the two isozymes indicated that the major isozyme was more susceptible to ATP and citrate inhibition as well as relief of ATP and citrate inhibition by fructose-6-P, AMP, and ammonia. With the use of DEAE-cellulose chromatography and antiserum titration of 100,000gsupernatant fluids, it was shown that the two hepatic isozymes were always found together in adult, embryonic, and neoplastic liver and in kidney.