Characterization of glycogen-synthase phosphatase and phosphorylase phosphatase in subcellular liver fractions.
Upon fractionation of a postmitochondrial supernatant from rat liver, the synthase phosphatase (EC 3.1.3.42) activity (assayed at high tissue concentrations) was largely recovered in the glycogen fraction and to a minor extent in the cytosol. In contrast, the phosphorylase phosphatase (EC 3.1.3.17) activity was approximately equally distributed between these two fractions, a lesser amount being recovered in the microsomal fraction. The phosphatase activities in the microsomal and glycogen fractions were almost completely inhibited by a preincubation with the modulator protein, a specific inhibitor of type-1 (ATP,Mg-dependent) protein phosphatases. In the cytosolic fraction, however, type-2A (polycation-stimulated) phosphatase(s) contributed significantly to the dephosphorylation of phosphorylase and of in vitro phosphorylated muscular synthase. Liver synthase b, used as substrate for the measurement of synthase phosphatase throughout this work, was only activated by modulator-sensitive phosphatases. Trypsin treatment of the subcellular fractions resulted in a dramatically increased (up to 1000-fold) sensitivity to modulator, a several-fold increase in phosphorylase phosphatase activity and a complete loss of synthase phosphatase activity. Similar changes occurred during dilution of the glycogen-bound enzyme. A preincubation with the deinhibitor protein, which is known to counteract the effects of inhibitor-1 and modulator, increased several-fold the phosphorylase phosphatase activity, but exclusively in the cytosolic and microsomal fractions. It did not affect the synthase phosphatase activity. Taken together, the results indicate the existence of distinct, multi-subunit type-1 phosphatases in the cytosolic, microsomal and glycogen fractions.
Bollen
M
M
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Belgium.
Vandenheede
J R
JR
Goris
J
J
Stalmans
W
W
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006007:Glycogen-Synthase-D Phosphatase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008112:Liver Glycogen / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000201:enzymology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010765:Phosphorylase Phosphatase / Q000378:metabolism*; D051381:Rats; D012996:Solutions; D013347:Subcellular Fractions / Q000201:enzymology
D008112:Liver Glycogen; D012996:Solutions; D010749:Phosphoprotein Phosphatases; D010765:Phosphorylase Phosphatase; D006007:Glycogen-Synthase-D Phosphatase