The Rigulation of Glycogen Metabolism

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Abstract
Inhibitor‐1 from rabbit skeletal muscle was phosphorylated by protein kinase dependent on adenosine 3′: 5′‐ monophosphate (cyclic AMP0, but not by phosphorylase kinase or by glycogen synthetase kinase‐2. Protein phosphatase‐III, isolated and stored in the presence of manganese ions to keep it stable, was in a form which catalysed a rapid dephosphorylation and inactivation of inhibitor‐1. the kinetic constants for the dephosphorylation of inhibitor‐1 [Km= 0.7 μM, V(rel)=40] were comparable to those for the dephosphorylation of phosphorylase kinase [Km = 1.1 μM, V(rel)= 62] and phosphorylase [Km = 5.0 μM, V(rel) = 100]. The dephosphorylation of inhibitor‐III, and not by another enzyme that might be contaminating the preparation. When protein phosphatase‐III was diluted into buffers containing excess EDTA. it lost activity initially, but after 90 min, the activity reached a plateau that remainned stable for at least 20 h. The initial loss in activity varied with the substrate that was tested; it was 20‐30% with phosphorylase a, 50–60% with phosphorylase kinase and ≥ 95% with inhibitor‐1. This form of protein phosphatase‐III was inhibited by inhibitor‐1 in a noncompetitive manner, and the Ki for inhibitor‐1 was 1.6 ± 0.3 nM. The phosphorylase phosphatase, phosphorylase kinase phosphatase and glycogen synthetase phosphatase activities of protein phosphatase‐III were inhibited in an identical manner by inhibiter‐1. This result emphasizes the potential importance of inhibitor‐1 in the regulation of glycogen metabolism, since it can influence the state of phosphorylation of three defferent enzymes. The formation of the inactive complex between inhibitor‐1 and protein phosphatase‐III was reversed by incubation with trypsin (which destroyed inhibitor‐1, but not ptotein phosphatase‐III) or by dilution of the inactive complex. Kinetic studies, using the from of protein phosphatase‐III which dephosphorylated inhibitor‐1 very rapidly, demonstrated three unusual features of the system: (a) inhibitor‐1 was still as powerful an inhibitor of the dephosphorylation of phosphorylase a and phosphorylase kinase a even under conditions where it was being rapidly dephosphorylated; (b) inhibitor‐1 was not an inhibitor of its own dephosphorylation; (c) phosphorylase a did not effect the rate of dephosphorylation of inhibitor‐1 even when it was present in a 50‐fold molar excess over inhibitor‐1. The result of these three properties is that inhibitor‐1 is preferentially dephosphorylated by prorein phosphatase‐III even in the presence of a large excess of other phosphoprotein substrates. Inhibitor‐1 was also dephosphorylated by protein phosphatase‐II. The kinetic constants for the dephosphotylation of inhibitor‐1 [Km = 2.8 μM, V(rel) = 200] and the α‐subunit of phosphorylase kinase[Km = 3.7 μM, V(rel) = 100] were comparable. Inhibitor‐1 only inhibited protein phosphatase‐II by virtue of its ability to aact as an alternative substrate for the enzyme, and it inhibited protein phosphatase‐II at least several hundred‐fold less effectively than protein phosphatase‐III. The dephosphorylated form of inhibitor‐1 did not affect he activity jof protein phosphatase‐II or protein phosphatase‐III, nor did it affect the ability of protein phosphatase‐III to be inhibited by the phosphorylated form of inhibitor‐1, even when present in a 50‐fold molar excess over the phosphorylated form. Inhibitor‐1 was partially purified by a procedure in which the initial heat treatment at 90 °C was omitted. The behaviour of the protein on ion‐exchange chromatography, gel filtration and gel electrophoresis was identical to that of the protein prepared from heated extracts, and activation of the preparation by phosphorylation with cyclic‐AMP‐dependent protein kinase was accompanied by the forination of phosphothreonine. The results showed that inhibitor‐1 was not an artefact produced by heating muscle extracts at 90 °C. The evidence which indicates that the dephosphorylated form of inhibitor‐1 and protein phosphatase‐III do not form a complex, and the possibility that protein phosphatase‐III is the enzyme which dephosphorylates inhibitor‐1 in vivo are discussed.