J. Biol. Chem., Vol. 259, Issue 14, 8718-8723, Jul, 1984
Purification and phosphorylation of fructose-1,6-bisphosphatase from Kluyveromyces fragilis
Y Toyoda and J Sy
Fructose-1,6-bisphosphatase from the yeast Kluyveromyces fragilis was found
to have an apparent Mr = 155,000 and to be composed of four Mr = 35,000
subunits. The extent and rate of phosphorylation of fructose-1,6-
bisphosphatase (Fru-1,6-P2) by yeast cAMP-dependent protein kinase were
dependent on fructose-1,6-bisphosphatase inhibitors, 5'-AMP and fructose
2,6-bisphosphate (Fru-2,6-P2). In the absence of inhibitor, the enzyme was
slowly phosphorylated with a maximum incorporation of 1 mol of
phosphate/mol of enzyme. The presence of both inhibitors greatly increased
the phosphorylation rate with a maximum incorporation of 2 mol of
phosphate/mol of enzyme. The presence of only one inhibitor led to an
intermediate rate of phosphorylation with 2 mol of phosphate
incorporated/mol of enzyme. There was no significant change in enzymatic
activity after phosphorylation. The estimated sedimentation coefficient of
fructose-1,6-bisphosphatase was lowered by 5'-AMP from 8.2 to 5.7 while
Fru-2,6-P2 increased the S value to 8.5. The presence of either Fru-1,6-P2
or Fru-2,6-P2 prevented the 5'-AMP lowering of S value. The susceptibility
of enzyme to partial tryptic digestion was not changed by the presence of
5'-AMP. The presence of both Fru-2,6-P2 and 5'-AMP led to the protection of
Mr = 35,000 subunit from tryptic digestion while Fru-2,6-P2 alone led to a
protection of an Mr = 30,000 peptide fragment. This peptide fragment did
not contain the phosphorylation sites. Our results suggest that the rapid
regulation of fructose-1,6-bisphosphatase following glucose addition is
controlled mainly by enzyme inhibitors.
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