title: Stabilization of the hexameric glutamate dehydrogenase from Escherichia coli by cations and polyethyleneimine.
abstract: The enzyme glutamate dehydrogenase (GDH) from Escherichia coli is a hexameric protein. The stability of this enzyme was increased in the presence of Li(+) in concentrations ranging from 1 to 10mM, 1M of sodium phosphate, or 1M ammonium sulfate. A very significant dependence of the enzyme stability on protein concentration was found, suggesting that subunit dissociation could be the first step of GDH inactivation. This effect of enzyme concentration on its stability was not significantly decreased by the presence of 10mM Li(+). Subunit crosslinking could not be performed using neither dextran nor glutaraldehyde because both reagents readily inactivated GDH. Thus, they were discarded as crosslinking reagents and GDH was incubated in the presence of polyethyleneimine (PEI) with the aim of physically crosslinking the enzyme subunits. This incubation does not have a significant effect on enzyme activity. However, after optimization, the PEI-GDH was found to almost maintain the full initial activity after 2h under conditions where the untreated enzyme retained only 20% of the initial activity, and the effect of the enzyme concentration on enzyme stability almost disappeared. This stabilization was maintained in the pH range 5-9, but it was lost at high ionic strength. This PEI-GDH composite was also much more stable than the unmodified enzyme in stirred systems. The results suggested that a real adsorption of the PEI on the GDH surface was required to obtain this stabilizing effect. A positive effect of Li(+) on enzyme stability was maintained after enzyme surface coating with PEI, suggesting that the effects of both stabilizing agents could not be exactly based on the same mechanism. Thus, the coating of GDH surface with PEI seems to be a good alternative to have a stabilized and soluble composite of the enzyme.
authors.affiliation: Departamento de Biocatálisis, ICP-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Garcia-Galan
authors.forename: Oveimar
authors.initials: O
authors.lastname: Barbosa
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernandez-Lafuente
mesh_terms: D002414:Cations, Monovalent / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D018086:Glutamate Dehydrogenase (NADP+) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000494:pharmacology; D008958:Models, Molecular; D011094:Polyethyleneimine / Q000494:pharmacology; D020836:Protein Structure, Quaternary
chemical_list: D002414:Cations, Monovalent; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D011094:Polyethyleneimine; D008094:Lithium; D018086:Glutamate Dehydrogenase (NADP+)