Tan A W, Nuttall F Q
J Biol Chem. 1985 Apr 25;260(8):4751-7.
Our previous study (Tan, A. W. H., and Nuttall, F. Q. (1983) J. Biol. Chem. 258, 9624-9630) indicated that liver synthase D contained a large number of endogenous phosphates, 12 of which were stable and 6 labile to alkali treatment. We wished to investigate the nature of the phosphates on synthase which became isotopically labeled when inorganic [32P]phosphate was given either to intact rats or to isolated liver cells. An antibody against liver synthase D was used for the isolation of synthase. The antibody recognized both the phosphorylated and dephosphorylated form of the enzyme, native as well as partially cleaved species. A large enzyme form, with Mr of 90,000 as well as one with Mr of 73,000 was observed. A 61% decrease in [32P]phosphate was found in synthase when prelabeled liver cells were treated with glucose, whereas a 25% increase was seen in cells treated with glucagon. After [32P]synthase D was converted to synthase I by synthase phosphatase, 95% of the [32P]phosphate was lost. All of the bound [32P]phosphates were found to be labile to alkali. Thus, under the in vivo conditions used, the [32P]phosphates incorporated into synthase were characterized by their fast turnover rate, alkali lability and susceptibility to the action of synthase phosphatase, both in vivo and in vitro. These criteria serve to distinguish them from the slower turning-over, alkali-stable phosphates found previously in both synthases D and I.
我们之前的研究（谭，A. W. H.，和纳托尔，F. Q.（1983年）《生物化学杂志》258卷，9624 - 9630页）表明，肝脏合酶D含有大量内源性磷酸盐，其中12个是稳定的，6个对碱处理不稳定。我们希望研究当向完整大鼠或分离的肝细胞给予无机[32P]磷酸盐时，合酶上那些被同位素标记的磷酸盐的性质。一种针对肝脏合酶D的抗体被用于分离合酶。该抗体能识别该酶的磷酸化和去磷酸化形式，包括天然形式以及部分裂解形式。观察到一种分子量为90,000的大酶形式以及一种分子量为73,000的酶形式。当用葡萄糖处理预先标记的肝细胞时，合酶中的[32P]磷酸盐减少了61%，而用胰高血糖素处理的细胞中则增加了25%。[32P]合酶D被合酶磷酸酶转化为合酶I后，95%的[32P]磷酸盐丢失了。所有结合的[32P]磷酸盐都被发现对碱不稳定。因此，在所使用的体内条件下，掺入合酶的[32P]磷酸盐的特点是其周转速度快、对碱不稳定以及在体内和体外都易受合酶磷酸酶作用的影响。这些标准有助于将它们与先前在合酶D和I中发现的周转较慢、对碱稳定的磷酸盐区分开来。
