Abstract
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.
Highlights
A heat-stable protein inhibitor of phosphoprotein phosphatase has been purified to homogeneity from rabbit liver extract by heating to 95” followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-ZOO
Phosphatase Inhibitor - Inhibitor activity was determined by its ability to inhibit the dephosphorylation of YPlphosphorylase a catalyzed by rabbit liver phosphoprotein phosphatase
Brandt et al [2], while studying the properties of a crude inhibitor preparation from rabbit liver, observed that gel filtration of inhibitor on a column of Bio-Gel A-0.5m resulted in elution of the activity with seemingly multiple molecular forms
Summary
A heat-stable protein inhibitor of phosphoprotein phosphatase has been purified to homogeneity from rabbit liver extract by heating to 95” followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-ZOO. A kinetic analysis of the effect of the inhibitor on the dephosphorylation of FPlphosphorylase 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 demonstrated in all rat tissues tested. It has been suggested that a heat-stable protein inhibitor of phosphoprotein phosphatase plays an important role in the regulation of this enzyme (l-5). The presence of this inhibitor was first observed by Brandt et al. The present communication reports the purification to homogeneity of a heat-stable protein inhibitor of phosphoprotein phosphatase from this tissue
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