title: Phosphorylation in vivo of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase at the cyclic AMP-dependent site.
abstract: 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.
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism*; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
chemical_list: D000242:Cyclic AMP; D006597:Fructose-Bisphosphatase; D014357:Trypsin