Abstract
The glycogen‐bound form of protein phosphatase‐1 (PP‐1g) was previously purified as a heterodimer composed of a 37‐kDa catalytic (C) subunit and a proteolytically sensitive 103‐kDa glycogen‐binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161‐kDa protein, and that the 103‐kDa species (now termed G′) is itself a product of proteolysis. A second phosphorylation site for cAMP‐dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP‐1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP‐1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen‐protein particles during washing with a variety of solvents. The PP‐1G holoenzyme was released from glycogen‐protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP‐1G. Binding experiments with purified PP‐1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high‐affinity glycogen‐binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP‐1G should be almost entirely bound to glycogen.
| Original language | English |
|---|---|
| Pages (from-to) | 457-465 |
| Number of pages | 9 |
| Journal | European Journal of Biochemistry |
| Volume | 180 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Dec 1989 |
Fingerprint
Cite this
}
The glycogen‐binding subunit of protein phosphatase‐1g from rabbit skeletal muscle : Further characterisation of its structure and glycogen‐binding properties. / Hubbard, Michael J.; Cohen, Philip.
In: European Journal of Biochemistry, Vol. 180, No. 2, 12.1989, p. 457-465.Research output: Contribution to journal › Article
TY - JOUR
T1 - The glycogen‐binding subunit of protein phosphatase‐1g from rabbit skeletal muscle
T2 - Further characterisation of its structure and glycogen‐binding properties
AU - Hubbard, Michael J.
AU - Cohen, Philip
PY - 1989/12
Y1 - 1989/12
N2 - The glycogen‐bound form of protein phosphatase‐1 (PP‐1g) was previously purified as a heterodimer composed of a 37‐kDa catalytic (C) subunit and a proteolytically sensitive 103‐kDa glycogen‐binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161‐kDa protein, and that the 103‐kDa species (now termed G′) is itself a product of proteolysis. A second phosphorylation site for cAMP‐dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP‐1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP‐1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen‐protein particles during washing with a variety of solvents. The PP‐1G holoenzyme was released from glycogen‐protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP‐1G. Binding experiments with purified PP‐1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high‐affinity glycogen‐binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP‐1G should be almost entirely bound to glycogen.
AB - The glycogen‐bound form of protein phosphatase‐1 (PP‐1g) was previously purified as a heterodimer composed of a 37‐kDa catalytic (C) subunit and a proteolytically sensitive 103‐kDa glycogen‐binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161‐kDa protein, and that the 103‐kDa species (now termed G′) is itself a product of proteolysis. A second phosphorylation site for cAMP‐dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP‐1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP‐1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen‐protein particles during washing with a variety of solvents. The PP‐1G holoenzyme was released from glycogen‐protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP‐1G. Binding experiments with purified PP‐1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high‐affinity glycogen‐binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP‐1G should be almost entirely bound to glycogen.
UR - http://www.scopus.com/inward/record.url?scp=0024603271&partnerID=8YFLogxK
U2 - 10.1111/j.1432-1033.1989.tb14668.x
DO - 10.1111/j.1432-1033.1989.tb14668.x
M3 - Article
VL - 180
SP - 457
EP - 465
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
SN - 0014-2956
IS - 2
ER -