Sakakibara R, Uyeda K
J Biol Chem. 1983 Jul 25;258(14):8656-62.
Low phosphate and high phosphate forms of phosphofructokinase (Furuya, E., and Uyeda, K. (1980) J. Biol. Chem. 255, 11656-11659) from rat liver were purified to homogeneity and various properties were compared. The specific activities of these enzymes and their electrophoretic mobilities on polyacrylamide in sodium dodecyl sulfate are the same. A limited tryptic digestion yields products with no change in the enzyme activity but with a reduction in the molecular weight of about 2000. Both low and high phosphate enzymes can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, and approximately twice as much [32P]phosphate is incorporated into the low phosphate than the high phosphate enzyme. A comparison of their allosteric kinetic properties reveal that the high phosphate enzyme is much more sensitive to inhibition by ATP and citrate and shows a higher K0.5 for fructose 6-phosphate than the low phosphate enzyme, and the difference in the K0.5 values becomes greater at lower pH values. Furthermore, the high phosphate phosphofructokinase is less sensitive to activation by AMP and fructose 2,6-bisphosphate. Moreover, when the low phosphate enzyme is phosphorylated by protein kinase, the resulting phosphorylated enzyme exhibits a higher K0.5 for fructose 2,6-bisphosphate than does the untreated enzyme. These results demonstrate that the phosphorylation affects the allosteric kinetic properties of the enzyme and results in a less active form of phosphofructokinase.
从大鼠肝脏中纯化出磷酸果糖激酶的低磷酸化和高磷酸化形式（古谷英树和上田健一，(1980) 《生物化学杂志》255, 11656 - 11659），并比较了它们的各种特性。这些酶的比活性以及它们在十二烷基硫酸钠聚丙烯酰胺凝胶上的电泳迁移率相同。有限的胰蛋白酶消化产生的产物，酶活性不变，但分子量降低了约2000。低磷酸化和高磷酸化的酶都可以被环磷酸腺苷依赖性蛋白激酶的催化亚基磷酸化，并且掺入低磷酸化酶中的[32P]磷酸大约是高磷酸化酶的两倍。对它们变构动力学特性的比较表明，高磷酸化酶对ATP和柠檬酸的抑制更为敏感，并且对6 - 磷酸果糖的K0.5值高于低磷酸化酶，在较低pH值下，K0.5值的差异变得更大。此外，高磷酸化的磷酸果糖激酶对AMP和2,6 - 二磷酸果糖的激活不太敏感。而且，当低磷酸化酶被蛋白激酶磷酸化时，所得的磷酸化酶对2,6 - 二磷酸果糖的K0.5值高于未处理的酶。这些结果表明，磷酸化影响了该酶的变构动力学特性，并导致磷酸果糖激酶形成活性较低的形式。
