Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle

Abstract

A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian cells.

A highly purified rat‐liver phosphoprotein phosphatase preparation with activity towards phosphopyruvate kinase (Type L)

“…No effect of these inhibitors was obtained at 5 PM [32P]pllospllopyruvate kinase, [32P]phosphoprotan~ine and [32P]phosphorylase a. These types of inhibitors are present in several tissues and are known to inhibit phosphorylase a phosphatase activity [21,22]. The cause of the lack of inhibition is not known at the present, but the findings suggest that not all enzymes with phosphorylase a phosphatase activity are inhibited by these inhibitors.…”

“…However, in contrast to other preparations, the present one tends to be destabilized when stored in EDTA and has so far proved to be insensitive to inhibition by heat-stable inhibitors 1 and 2 prepared from rabbit skeletal muscle [21,34]. No effect of these inhibitors was obtained at 5 PM [32P]pllospllopyruvate kinase, [32P]phosphoprotan~ine and [32P]phosphorylase a.…”

The Protein Phosphatases Involved in Cellular Regulation. 1. Classification and Substrate Specificities

“…Inhibitor-I was only an inhibitor after it had been phosphorylated by cyclic-AMP-dependent protein kinase [14]. Neither inhibitor-I nor inhibitor-2 affected the activity of protein phosphatase-2 [15,16].While our own studies were in progress, Lee and coworkers purified a z 35 OOO-M, phosphorylase phosphatase from rabbit liver using an unusual procedure involving 80 "/:ethanol precipitation at room temperature [19]. This procedure not only denatured many contaminating proteins, but also appeared to dissociate the z 35000-M, catalytic subunit from higher M , complexes, containing other subunits [20].…”

“…Protein phosphatase-I was subsequently shown to be the major protein phosphatase acting on glycogen synthase and glycogen phosphorylase in skeletal muscle, and was therefore responsible for all the dephosphorylation reactions that inhibited glycogenolysis or activated glycogen synthesis [8,12,131. In contrast, protein phosphatase-2 only accounted for a minor proportion of the glycogen synthase phosphatase and phosphorylase phosphatase activity in skeletal muscle [12,13].Protein phosphatase-I was shown to be inhibited by nanomolar concentrations of two heat-stable proteins in skeletal muscle, termed inhibitor-I and inhibitor-2 [12,[14][15][16], which were purified to homogeneity and characterised extensively [17,18]. Inhibitor-I was only an inhibitor after it had been phosphorylated by cyclic-AMP-dependent protein kinase [14].…”

“…Protein phosphatase-I was shown to be inhibited by nanomolar concentrations of two heat-stable proteins in skeletal muscle, termed inhibitor-I and inhibitor-2 [12,[14][15][16], which were purified to homogeneity and characterised extensively [17,18]. Inhibitor-I was only an inhibitor after it had been phosphorylated by cyclic-AMP-dependent protein kinase [14].…”