Purification and characterization of a divalent cation-independent, spermine-stimulated protein phosphatase from bovine kidney mitochondria.
A divalent cation-independent and spermine-stimulated phosphatase (protein phosphatase SP) that is active toward the phosphorylated pyruvate dehydrogenase complex has been purified about 15,000-fold to near homogeneity from extracts of bovine kidney mitochondria. Half-maximal stimulation, 1.5- to 3-fold at pH 7.0-7.3, occurred at 0.5 mM spermine. Protein phosphatase SP exhibited an apparent Mr = 140,000-170,000 as estimated by gel-filtration chromatography on Sephacryl S-300. Two major subunits, with apparent Mr = 60,000 and 34,000, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel-permeation chromatography of protein phosphatase SP on Sephacryl S-200 in the presence of 6 M urea and 1.4 M NaCl increased its activity 3- to 6-fold and was accompanied by conversion to the catalytic subunit with an apparent Mr = approximately 34,000. Protein phosphatase SP was inactive with p-nitrophenyl phosphate and was not inhibited by protein phosphatase inhibitor 1, inhibitor 2, or the protein inhibitor of branched-chain alpha-keto acid dehydrogenase phosphatase. Protein phosphatase SP was inhibited by sheep antibody to the catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle. It appears that protein phosphatase SP is related to protein phosphatase 2A.
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D007668:Kidney / Q000201:enzymology / Q000648:ultrastructure*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011402:Pronase / Q000378:metabolism; D054648:Protein Phosphatase 2; D013096:Spermine / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism
D002413:Cations, Divalent; D009711:Nucleotides; D010446:Peptide Fragments; D013096:Spermine; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D014357:Trypsin; D011402:Pronase