Sheorain V S, Corbin J D, Soderling T R
J Biol Chem. 1985 Feb 10;260(3):1567-72.
Rabbit skeletal muscle glycogen synthase (synthase a) can be phosphorylated by 0.2 to 2.5 microM catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.5 to 3 mol of 32PO4/subunit (90,000 X g). When a complete tryptic digest of this 32P-synthase was chromatographed on reverse phase high performance liquid chromatography, it was observed that, in addition to sites 1a, 1b, and 2, site 3 was phosphorylated. The peptide containing site 5 also contained 32PO4, but sequence analysis identified a new phosphorylation site, site 4 (Arg-His-Ser-Ser(PO4)-) which precedes site 5. Phosphorylation of sites 3 and 4 became significant when the total phosphorylation stoichiometry exceeded 1.5 mol/subunit. The heat-stable protein kinase inhibitor protein or the regulatory subunit of cAMP-dependent protein kinase decreased the phosphorylation of all sites, including sites 3 and 4. Phosphorylation of all sites by the holoenzyme form of cAMP-dependent kinase was highly dependent on the presence of cAMP. These results establish that phosphorylation of these sites is due to the cAMP-dependent protein kinase itself and not to a contaminating kinase(s). Synthase b from control rabbit muscle, containing 2 to 2.5 mol of phosphate/subunit, incorporated up to 2.0 mol of 32PO4 catalyzed in vitro by the cAMP-dependent protein kinase. Again, significant 32PO4 was detected in sites 3 and 4 as well as in 1a, 1b, and 2. These results suggest that the in vivo phosphorylation of sites 1a, 1b, 2, and 3 observed after injection of epinephrine in rabbits could be due solely to the known activation of the cAMP-dependent protein kinase.
兔骨骼肌糖原合酶（合酶a）可被0.2至2.5微摩尔的环磷酸腺苷（cAMP）依赖性蛋白激酶催化亚基磷酸化，化学计量比为1.5至3摩尔的32P-磷酸/亚基（90,000×g）。当将这种32P-合酶的完整胰蛋白酶消化产物在反相高效液相色谱上进行层析时，观察到除了位点1a、1b和2外，位点3也被磷酸化。包含位点5的肽也含有32P-磷酸，但序列分析确定了一个新的磷酸化位点，即在位点5之前的位点4（精氨酸-组氨酸-丝氨酸-丝氨酸（磷酸化）-）。当总磷酸化化学计量比超过1.5摩尔/亚基时，位点3和4的磷酸化变得显著。热稳定蛋白激酶抑制蛋白或cAMP依赖性蛋白激酶的调节亚基降低了所有位点的磷酸化，包括位点3和4。cAMP依赖性激酶全酶形式对所有位点的磷酸化高度依赖于cAMP的存在。这些结果表明，这些位点的磷酸化是由于cAMP依赖性蛋白激酶本身，而不是由于污染的激酶。来自对照兔肌肉的合酶b，每个亚基含有2至2.5摩尔的磷酸盐，在体外被cAMP依赖性蛋白激酶催化掺入多达2.0摩尔的32P-磷酸。同样，在位点3和4以及1a、1b和2中检测到显著的32P-磷酸。这些结果表明，在给兔子注射肾上腺素后体内观察到的位点1a、1b、2和3的磷酸化可能仅归因于cAMP依赖性蛋白激酶的已知激活。
