Affinity chromatography of regulatory subunits of protein phosphatase-1.
In this study we demonstrate that recombinant rabbit muscle protein phosphatase-1 immobilized on CH-Sepharose is an efficient affinity chromatography support for the isolation of subunits of phosphatase-1. The support was used to isolate the glycogen binding subunit of phosphatase-1 from muscle and nonmuscle rat tissues. Examination of the affinity-purified material from rat heart and liver showed that the major component was a 160-kDa polypeptide on SDS-PAGE. The identity of the purified liver 160-kDa polypeptide as the glycogen binding subunit was confirmed by the demonstration that it is capable of binding to glycogen, and is phosphorylated by the catalytic subunit of PKA. The novel observation was made that the phosphorylation was dependent on the presence of glycogen. Examination of the material from heart, lung, liver, kidney, and brain showed a similar phenomenon. Our studies show that this subunit is widely distributed in tissues. The affinity support was also efficient in the isolation of the NIPP-1 (nuclear inhibitor of protein phosphatase-1) proteins from calf thymus. Examination of heat-treated extracts of rat liver led to the isolation of a novel 19-kDa inhibitory protein which could also be phosphorylated by PKA and may represent the rat liver homolog of calf thymus NIPP-1.
Zhao
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Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Xia
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Lee
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D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D004800:Enzymes, Immobilized; D006003:Glycogen / Q000378:metabolism; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009928:Organ Specificity; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D011506:Proteins / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D013950:Thymus Gland / Q000737:chemistry
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C021042:protein phosphatase inhibitor-1; D006003:Glycogen; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1