Incubation of purified skeletal muscle glycogen synthetase I with trypsin (10 μg/ml) for 15 min decreased the Stokes radius of the enzyme from 68 Å to 62 Å and the subunit molecular weight from 90,000 to about 73,000. No decrease in the sedimentation coefficient of 13.3 S could be detected. It was calculated that native synthetase I is a tetramer of molecular weight 360,000 to 370.000. Trypsin also catalyzed a decrease in the synthetase activity ratio (minus glucose 6 P to plus glucose 6 P) largely by reducing minus glucose 6 P activity. The magnitude of the trypsin effect on the synthetase activity ratio was very similar to that produced by phosphorylation of synthetase by the cyclic AMP dependent protein kinase. The activity ratio could be lowered from that characteristic of synthetase I, 0.85, to 0.25 either by trypsin digestion or by incorporation of 1 mol of P1 per mol of synthetase subunit. An activity ratio of <0.05 could be obtained in three ways as follows, (a) phosphorylation to 2 P1/subunit, (b) phosphorylation to 1 P1/subunit (ratio = 0.25) followed by trypsin treatment, (c) trypsin treatment (ratio = 0.25) followed by phosphorylation. When trypsinized synthetase was phosphorylated by the catalytic subunit of cyclic AMP dependent protein kinase, 1 P1/subunit was incorporated. Trypsin (6 μg/ml) led to a rapid release of about 50% of the radioactivity from 32P synthetase regardless of whether the enzyme contained 1 or 2 phosphates per subunit. It was concluded that two sites on the enzyme subunit are highly susceptible to phosphorylation catalyzed by the cyclic AMP dependent protein kinase. The data indicate that the 1st mol of P1 incorporated is distributed about equally between the two sites. A model is proposed to account for these observations. The data further indicate that a peptide containing one site is removed by trypsin. This reduces enzyme activity to the same extent as does phosphorylation of the site in the intact protein.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1976|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology