Rittenhouse J, Moberly L, Marcus F
J Biol Chem. 1987 Jul 25;262(21):10114-9.
In vivo labeled fructose-1,6-bisphosphatase was immunopurified from yeast (Saccharomyces cerevisiae) cells that had been incubated in the presence of [32P] orthophosphate. Tryptic peptides from labeled enzyme were mapped by high performance liquid chromatography. Most of the radioactivity was found to be associated with the peptide Arg9 through Arg24, the same peptide which had been previously shown to be phosphorylated in vitro by cAMP-dependent protein kinase (Rittenhouse, J., Harrsch, P. B., Kim, J. N., and Marcus, F. (1986) J. Biol. Chem. 261, 3939-3943). The amino acid sequence analysis suggests that phosphorylation occurs at the same site, Ser11. We have also determined the extent of phosphorylation at Ser11 of fructose-1,6-bisphosphatase in yeast cultures growing under various nutritional conditions by measuring the relative amounts of phospho- and corresponding dephosphopeptides in tryptic digests. Significant levels of phosphorylation of the enzyme were found in yeast cultures grown under gluconeogenic conditions that varied from 0.15 to 0.50 mol of phosphate per mol of enzyme subunit. However, phosphate incorporation rapidly increased to greater than 0.8 mol after addition of glucose to these cultures. An alternative technique, based solely on enzyme activity measurements, was also developed to estimate the extent of fructose-1,6-bisphosphatase phosphorylation in yeast cultures. The results obtained with this technique agreed with those obtained by high performance liquid chromatography of tryptic peptides.
从在[32P]正磷酸盐存在下孵育的酵母（酿酒酵母）细胞中免疫纯化体内标记的果糖-1,6-二磷酸酶。通过高效液相色谱法对标记酶的胰蛋白酶肽进行图谱分析。发现大部分放射性与肽Arg9至Arg24相关，该肽先前已显示在体外被cAMP依赖性蛋白激酶磷酸化（Rittenhouse, J., Harrsch, P. B., Kim, J. N., and Marcus, F. (1986) J. Biol. Chem. 261, 3939 - 3943）。氨基酸序列分析表明磷酸化发生在同一位点，即Ser11。我们还通过测量胰蛋白酶消化物中磷酸化和相应去磷酸化肽的相对量，确定了在各种营养条件下生长的酵母培养物中果糖-1,6-二磷酸酶Ser11位点的磷酸化程度。在糖异生条件下生长的酵母培养物中发现该酶有显著水平的磷酸化，每摩尔酶亚基的磷酸含量为0.15至0.50摩尔。然而，向这些培养物中添加葡萄糖后，磷酸盐掺入量迅速增加至大于0.8摩尔。还开发了一种仅基于酶活性测量的替代技术，以估计酵母培养物中果糖-1,6-二磷酸酶的磷酸化程度。用该技术获得的结果与通过胰蛋白酶肽的高效液相色谱法获得的结果一致。
