ABSTRACT
The chromatography of rat liver crude extract (10 000 x g supernatant) on a Biogel A-0.5 m column resulted in two peaks of phosphorylase phosphatase activity. The first peak, eluted in void volume, is of microsomal origin whereas the second peak, a cytoplasmic origin, was eluted with an apparent molecular weight of 260 000. In the presence of NaCl, the cytosolic enzyme was dissociated into intermediary (Mr = 135 000 and 68 000) and low molecular weight (Mr = 35 000) forms with an apparent activation of the enzyme activity. Similar dissociation was observed in the presence of KCl and (NH4)2SO4. A partially purified high molecular weight phosphorylase phosphatase was also dissociated into the low molecular weight form by 0.2 M NaCl. However, a protein phosphatase showing activity towards 32P-labelled histone was not dissociated by 0.2 M NaCl or 0.1 M (NH4)2SO4.
Subject(s)
Liver/enzymology , Phosphoprotein Phosphatases/isolation & purification , Phosphorylase Phosphatase/isolation & purification , Animals , Molecular Weight , Phosphorylase Phosphatase/metabolism , Protein Conformation , Rats , Sodium ChlorideSubject(s)
Diabetes Mellitus, Experimental/metabolism , Glycogen/metabolism , Kidney/metabolism , Liver/metabolism , Animals , Blood Glucose/metabolism , Glucosephosphates/metabolism , Glycogen Synthase/metabolism , Insulin/pharmacology , Liver Glycogen/metabolism , Male , Phosphorylases/metabolism , RatsABSTRACT
A heat-stable protein inhibitor of phosphoprotein phosphatase has been purified to homogeneity from rabbit liver extract by heating to 95 degrees followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The purified inhibitor showed a single band when examined by gel electrophoresis S20, w and Stokes radius values were 1.45 and 25.5, respectively. Using these two values, the molecular weight and frictional ratio was calculated to be 15,500 and 3.40, respectively. The molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was found to be 14,200. The inhibition of phosphoprotein phosphatase was linear up to 40% inhibition with respect to inhibitor was constant with time of incubation for at least 30 min. The optimum pH for the inhibition was between 6.8 and 7.6. A kinetic analysis of the effect of the inhibitor on the dephosphorylation of [32P]phosphorylase a by rabbit liver phosphoprotein phosphatase indicated a noncompetitive inhibition with respect to phosphorylase a. Purified liver inhibitor inhibited the phosphoprotein phosphatase activity in all rat tissues examined. Utilizing purified rabbit liver phosphoprotein phosphatase, the presence of inhibitor activity was also demonstrated in all rat tissues tested.
Subject(s)
Liver/physiology , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphorylase Phosphatase/antagonists & inhibitors , Proteins/physiology , Animals , Drug Stability , Hot Temperature , Kinetics , Liver/enzymology , Proteins/isolation & purification , RabbitsABSTRACT
The effects of streptozotocin-induced diabetes and of insulin supplementation to diabetic rats on glycogen-metabolizing enzymes in liver were determined. The results were compared with those from control animals. The activities of glycogenolytic enzymes, i.e. phosphorylase (both a and b), phosphorylase kinase and protein kinase (in the presence or in the absence of cyclic AMP), were significantly decreased in the diabetic animals. The enzyme activities were restored to control values by insulin therapy. Glycogen synthase (I-form) activity, similarly decreased in the diabetic animals, was also restored to control values after the administration of insulin. The increase in glycogen synthase(I-form) activity after insulin treatment was associated with a concomitant increase in phosphoprotein phosphatase activity. The increase in phosphatase activity was due to (i) a change in the activity of the enzyme itself and (ii) a decrease in a heat stable protein inhibitor of the phosphatase activity.
