title: Purification and characterization of two potent heat-stable protein inhibitors of protein phosphatase 2A from bovine kidney.
abstract: Two heat-stable protein inhibitors of protein phosphatase 2A (PP2A), tentatively designated I1PP2A and I2PP2A, have been purified to apparent homogeneity from extracts of bovine kidney. The purified preparations of I1PP2A exhibited an apparent M(r) approximately 30,000 and 250,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-300, respectively. In contrast, the purified preparations of I2PP2A exhibited an apparent M(r) approximately 20,000 and 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200, respectively. The purified preparations of I1PP2A and I2PP2A inhibited PP2A with 32P-labeled myelin basic protein, 32P-labeled histone H1, 32P-labeled pyruvate dehydrogenase complex, 32P-labeled phosphorylase, and protamine kinase as substrates. By contrast, I1PP2A and I2PP2A exhibited little effect, if any, on the activity of PP2A with 32P-labeled casein, and did not prevent the autodephosphorylation of PP2A in incubations with the autophosphorylation-activated protein kinase [Guo, H., & Damuni, Z. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504]. The purified preparations of I1PP2A and I2PP2A had little effect, if any, on the activities of protein phosphatase 1, protein phosphatase 2B, protein phosphatase 2C, and pyruvate dehydrogenase phosphatase. With 32P-labeled MBP as a substrate, kinetic analysis according to Henderson showed that I1PP2A and I2PP2A were noncompetitive and displayed a Ki of about 30 and 25 nM, respectively. Following cleavage with Staphylococcus aureus V8 protease, I1PP2A and I2PP2A displayed distinct peptide patterns, indicating that these inhibitor proteins are the products of distinct genes. The N-terminal amino acid sequences of the purified preparations indicate that I1PP2A and I2PP2A are novel proteins.
authors.affiliation: Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey 17033.
authors.forename: M
authors.initials: M
authors.lastname: Li
authors.forename: H
authors.initials: H
authors.lastname: Guo
authors.forename: Z
authors.initials: Z
authors.lastname: Damuni
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D056488:Histone Chaperones; D006657:Histones / Q000378:metabolism; D006358:Hot Temperature; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000378:metabolism; D009687:Nuclear Proteins; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D016601:RNA-Binding Proteins; D014157:Transcription Factors
chemical_list: C087425:ANP32A protein, human; D002364:Caseins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D004676:Myelin Basic Protein; D009687:Nuclear Proteins; D010761:Phosphorus Radioisotopes; D011506:Proteins; D011768:Pyruvate Dehydrogenase Complex; D016601:RNA-Binding Proteins; C513473:SET protein, human; D014157:Transcription Factors; D006656:Protamine Kinase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2