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dc.contributor.authorKlarlund, Jes K.
dc.contributor.authorBradford, Andrew P.
dc.contributor.authorMilla, Maria G.
dc.contributor.authorCzech, Michael P.
dc.date2022-08-11T08:10:05.000
dc.date.accessioned2022-08-23T16:54:35Z
dc.date.available2022-08-23T16:54:35Z
dc.date.issued1990-01-05
dc.date.submitted2008-08-15
dc.identifier.citationJ Biol Chem. 1990 Jan 5;265(1):227-34.
dc.identifier.issn0021-9258 (Print)
dc.identifier.pmid2403557
dc.identifier.urihttp://hdl.handle.net/20.500.14038/42534
dc.description.abstractWe previously described a novel insulin-stimulated protein kinase activity that phosphorylates Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in cytosolic extracts of adipocytes (Yu, K-T., Khalaf, N., and Czech, M. P. (1987) J. Biol. Chem. 262, 16677-16685). In the present experiments, cytosolic extracts of livers from insulin-treated rats also exhibited a 30-100% increase in this Kemptide kinase activity and served as an abundant source for purification. The Kemptide kinase was purified in parallel from liver extracts of insulin-treated or control rats through five chromatographic steps and one polyethylene glycol precipitation. The chromatographic behavior of the insulin-stimulated Kemptide kinase differed significantly from the control kinase on Mono Q and heparin-Sepharose resins. The purified kinase preparations retain insulin stimulations of 2-10-fold. Analysis of the purified control and insulin-stimulated kinases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed single bands with similar silver staining intensity and apparent molecular masses of 52 kDa. The insulin-stimulated Kemptide phosphorylating activity also coincided with the major silver-stained band following isoelectric focusing in polyacrylamide gels. The stimulation of kinase activity in response to administration of insulin is due to an increase in Vmax, whereas the Km for Kemptide (0.3 mM) is unchanged. The apparent molecular mass of the native kinase determined by gel filtration is approximately 50 kDa, suggesting that it exists as a monomer. Either Mg2+ or Mn2+ serve as cofactors for the kinase which phosphorylates a variety of basic substrates including a number of peptides and histones. The activity of the Kemptide kinase is not changed by several compounds that have been shown to modulate other kinases. Based on these data, we conclude 1) a novel insulin-sensitive Kemptide kinase in liver cytosol has been purified to near homogeneity, and 2) insulin administration acutely modulates the specific activity of this Kemptide kinase in livers of intact rats.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=2403557&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttp://www.jbc.org/content/265/1/227.full.pdf+html
dc.subjectAnimals
dc.subjectChromatography
dc.subjectCytosol
dc.subjectElectrophoresis, Polyacrylamide Gel
dc.subjectInsulin
dc.subjectIsoelectric Focusing
dc.subjectLiver
dc.subjectMagnesium
dc.subjectMale
dc.subjectManganese
dc.subjectMolecular Weight
dc.subjectOligopeptides
dc.subjectProtein Kinases
dc.subjectRats
dc.subjectRats, Inbred Strains
dc.subjectSubstrate Specificity
dc.subjectLife Sciences
dc.subjectMedicine and Health Sciences
dc.titlePurification of a novel insulin-stimulated protein kinase from rat liver
dc.typeJournal Article
dc.source.journaltitleThe Journal of biological chemistry
dc.source.volume265
dc.source.issue1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/874
dc.identifier.contextkey579762
html.description.abstract<p>We previously described a novel insulin-stimulated protein kinase activity that phosphorylates Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in cytosolic extracts of adipocytes (Yu, K-T., Khalaf, N., and Czech, M. P. (1987) J. Biol. Chem. 262, 16677-16685). In the present experiments, cytosolic extracts of livers from insulin-treated rats also exhibited a 30-100% increase in this Kemptide kinase activity and served as an abundant source for purification. The Kemptide kinase was purified in parallel from liver extracts of insulin-treated or control rats through five chromatographic steps and one polyethylene glycol precipitation. The chromatographic behavior of the insulin-stimulated Kemptide kinase differed significantly from the control kinase on Mono Q and heparin-Sepharose resins. The purified kinase preparations retain insulin stimulations of 2-10-fold. Analysis of the purified control and insulin-stimulated kinases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed single bands with similar silver staining intensity and apparent molecular masses of 52 kDa. The insulin-stimulated Kemptide phosphorylating activity also coincided with the major silver-stained band following isoelectric focusing in polyacrylamide gels. The stimulation of kinase activity in response to administration of insulin is due to an increase in Vmax, whereas the Km for Kemptide (0.3 mM) is unchanged. The apparent molecular mass of the native kinase determined by gel filtration is approximately 50 kDa, suggesting that it exists as a monomer. Either Mg2+ or Mn2+ serve as cofactors for the kinase which phosphorylates a variety of basic substrates including a number of peptides and histones. The activity of the Kemptide kinase is not changed by several compounds that have been shown to modulate other kinases. Based on these data, we conclude 1) a novel insulin-sensitive Kemptide kinase in liver cytosol has been purified to near homogeneity, and 2) insulin administration acutely modulates the specific activity of this Kemptide kinase in livers of intact rats.</p>
dc.identifier.submissionpathoapubs/874
dc.contributor.departmentProgram of Molecular Medicine
dc.source.pages227-34


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