Sheorain V S, Juhl H, Bass M, Soderling T R
J Biol Chem. 1984 Jun 10;259(11):7024-30.
Phosphorylation site stoichiometries were determined for skeletal muscle glycogen synthase purified from control, alloxan-diabetic, and epinephrine-treated rabbits. One method of analysis was direct determination of the total in vivo phosphate content of each site after reverse phase high performance liquid chromatography separation of a complete tryptic digest of the purified synthase. The second method of analysis, in vitro phosphorylation, was based on the premise that in vitro 32P incorporation into each site would be inversely related to the in vivo phosphate content of that site. Glycogen synthase from control rabbits had the following distribution of in vivo phosphate (mole of phosphate/mol of site): site 1a, 0.29 +/- 0.08; site 5, 0.62 +/- 0.07; site 3, 0.46 +/- 0.06; site 1b, 0.23 +/- 0.03; and site 2, 0.43 +/- 0.07. Synthase from diabetic rabbits had 2-fold elevations of in vivo phosphate contents of sites 2 and 3. Epinephrine resulted in increased phosphorylation in vivo of site 1b (2.0-fold), site 2 (2.0-fold), and site 3 (1.5-fold). The in vitro phosphorylation analysis showed decreased 32P incorporation in vitro (indicative of increased in vivo phosphorylation) as follows: epinephrine, site 1a, site 3, site 1b, site 2; diabetic, site 3, site 2. The effect of diabetes on the in vitro phosphorylation of sites 2 and 3 was reversed by insulin treatment. We conclude that the major effect of epinephrine, phosphorylation of sites 1a, 1b, and 2, is mediated by the activation of the cAMP-dependent kinase. The mechanisms accounting for the phosphorylation of site 3 in response to epinephrine and phosphorylation of sites 2 and 3 in the diabetic state are under investigation.
测定了从对照、四氧嘧啶糖尿病和肾上腺素处理的兔子中纯化的骨骼肌糖原合酶的磷酸化位点化学计量。一种分析方法是在对纯化的合酶进行完整的胰蛋白酶消化产物进行反相高效液相色谱分离后，直接测定每个位点的体内总磷含量。第二种分析方法是体外磷酸化，其前提是体外将32P掺入每个位点与该位点的体内磷含量呈负相关。对照兔子的糖原合酶具有以下体内磷分布（磷摩尔数/位点摩尔数）：位点1a，0.29±0.08；位点5，0.62±0.07；位点3，0.46±0.06；位点1b，0.23±0.03；位点2，0.43±0.07。糖尿病兔子的合酶位点2和3的体内磷含量升高了2倍。肾上腺素导致位点1b（2.0倍）、位点2（2.0倍）和位点3（1.5倍）的体内磷酸化增加。体外磷酸化分析显示体外32P掺入减少（表明体内磷酸化增加）如下：肾上腺素，位点1a、位点3、位点1b、位点2；糖尿病，位点3、位点2。胰岛素治疗可逆转糖尿病对位点2和3体外磷酸化的影响。我们得出结论，肾上腺素的主要作用，即位点1a、1b和2的磷酸化，是由cAMP依赖性激酶的激活介导的。肾上腺素引起的位点3磷酸化以及糖尿病状态下位点2和3磷酸化的机制正在研究中。
