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Free keywords:
Alcohol Oxidoreductases/*metabolism
Bacterial Proteins/*metabolism
Chaperonin 60
Electrophoresis, Polyacrylamide Gel
Heat-Shock Proteins/*metabolism
Kinetics
Microbodies/*enzymology
Nucleotides/metabolism
Pichia/enzymology
Protein Binding
Abstract:
We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL. Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition of GroEL. Formation of complexes between GroEL and denatured AO was demonstrated by a gel-shift assay using non-denaturing polyacrylamide gel electrophoresis, and quantified by laser-densitometry of the gels. In the presence of MgAMP-PNP or MgADP the affinity of GroEL for AO was enhanced. Under these conditions up to 70% of the purified GroEL formed a complex with this protein. Release was stimulated at room temperature by MgATP, and was further enhanced by addition of GroES.
