High-affinity binding of glycogen-synthase phosphatase to glycogen particles in the liver. Role of glycogen in the inhibition of synthase phosphatase by phosphorylase a.
1. Post-mitochondrial supernatants were prepared from the livers of 24 h-fasted rats. Upon centrifugation at high speed, the major part of the glycogen-synthase phosphatase activity sedimented with the microsomal fraction. However, two approaches showed that the enzyme was associated with residual glycogen rather than with vesicles of the endoplasmic reticulum. Indeed, the activity was entirely solubilized when the remaining glycogen was degraded either by glucagon treatment in vivo or by alpha-amylolysis in vitro. No evidence could be found for an association of glycogen-synthase phosphatase with the smooth endoplasmic reticulum, as isolated with the use of discontinuous sucrose gradients. 2. After solubilization by glucagon treatment in vivo, synthase phosphatase could be transferred to glycogen particles with very high affinity. Half-maximal binding occurred at a glycogen concentration of about 0.25 mg/ml, whereas glycogen synthase and phosphorylase required 1.5-2 mg/ml. 3. In gel-filtered extracts prepared from glycogen-depleted livers, the activation of glycogen synthase was not inhibited at all by phosphorylase alpha. The inhibition was restored when the liver homogenates were prepared in a glycogen-containing buffer. The effect was half-maximal at a glycogen concentration of about 0.25 mg/ml, and virtually complete at 1 mg/ml. These findings explain long-standing observations that in fasted animals the liver contains appreciable amounts of both synthase and phosphorylase in the active form.
Mvumbi
L
L
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Belgium.
Stalmans
W
W
D000818:Animals; D002499:Centrifugation, Density Gradient; D004789:Enzyme Activation; D006003:Glycogen / Q000494:pharmacology; D006007:Glycogen-Synthase-D Phosphatase / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008112:Liver Glycogen / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D009844:Oligosaccharides / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010762:Phosphorylase a / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D008112:Liver Glycogen; D046911:Macromolecular Substances; D009844:Oligosaccharides; C030114:Aplanin; D006003:Glycogen; D010762:Phosphorylase a; D010749:Phosphoprotein Phosphatases; D006007:Glycogen-Synthase-D Phosphatase; D000516:alpha-Amylases