title: A native electrophoretic technique to study oligomerization and activity of cytosolic 5′-nucleotidase II.
abstract: The analysis of the oligomeric active state of a native protein usually requires the application of at least two analytical methods such as gel filtration and analytical ultracentrifugation. Both methods require a substantial amount of protein, time and/or expensive equipment. We here describe a native electrophoretic method for the identification of the native molecular weight of the recombinant wild-type cytosolic 5′-nucleotidase (cN-II) and of its mutants in subunit interfaces Y115A, F36R, K311A and G319Q. The protein was stained both with protein dye and with an activity staining method. Our results demonstrated that purified recombinant protein preparations contained substantial amounts of nucleic acids and misfolded, inactive protein. Furthermore, cN-II mutants K311A and G319Q in subunit interface assume a quaternary dimeric active form, while the only active quaternary structure of wild-type cN-II is the tetramer.
authors.forename: Daniela Nicole
authors.initials: DN
authors.lastname: Filoni
authors.forename: Rossana
authors.initials: R
authors.lastname: Pesi
authors.forename: Simone
authors.initials: S
authors.lastname: Allegrini
authors.forename: Marcella
authors.initials: M
authors.lastname: Camici
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Tozzi
mesh_terms: D015720:5'-Nucleotidase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D004247:DNA / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D004247:DNA; D015720:5'-Nucleotidase