Micellar electrokinetic chromatography as a complementary method to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for studying limited proteolysis of proteins.
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.
Viglio
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S
Dipartimento di Biochimica A. Castellani, Università di Pavia, Italy.
Valentini
G
G
Chiarelli
L
L
Zanaboni
G
G
Cetta
G
G
Iadarola
P
P
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
D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D012697:Serine Endopeptidases; D019272:Leukocyte Elastase; D014357:Trypsin; D020860:Subtilisin