title: Micellar electrokinetic chromatography as a complementary method to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for studying limited proteolysis of proteins.
abstract: Micellar electrokinetic chromatography (MEKC) has been utilized as an analytical method to perform investigations on limited proteolysis of proteins. To this purpose partial proteolysis experiments with a series of proteinases were performed, utilizing as model protein pyruvate kinase (PK) from Escherichia coli, an enzyme that is regulated allosterically by fructose 1,6-bisphosphate (FBP). Data obtained with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MEKC were compared; the profiles generated by submitting digests of PK treated with different proteinases in the presence and absence of FBP to electrophoretic analysis provided a useful adjunct for a better understanding of the effects of the allosteric activator on the conformation of the model enzyme. MEKC was also found to be a convenient technique for determining the kinetics of substrate proteolysis.
authors.affiliation: Dipartimento di Biochimica A. Castellani, Università di Pavia, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Viglio
authors.forename: G
authors.initials: G
authors.lastname: Valentini
authors.forename: L
authors.initials: L
authors.lastname: Chiarelli
authors.forename: G
authors.initials: G
authors.lastname: Zanaboni
authors.forename: G
authors.initials: G
authors.lastname: Cetta
authors.forename: P
authors.initials: P
authors.lastname: Iadarola
mesh_terms: D000818:Animals; D002417:Cattle; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D020860:Subtilisin; D013552:Swine; D014357:Trypsin / Q000378:metabolism
chemical_list: D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D012697:Serine Endopeptidases; D019272:Leukocyte Elastase; D014357:Trypsin; D020860:Subtilisin