Formation and quantification of protein complexes between peroxisomal alcohol oxidase and GroEL.
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.
Laboratory for Electron Microscopy, University of Groningen, The Netherlands.
M E
ME
Evers
T
T
Langer
W
W
Harder
F U
FU
Hartl
M
M
Veenhuis
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D018834:Chaperonin 60; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008830:Microbodies / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism; D010843:Pichia / Q000201:enzymology; D011485:Protein Binding
D001426:Bacterial Proteins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D009711:Nucleotides; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase