In vitro dissociation and re-assembly of peroxisomal alcohol oxidases of Hansenula polymorpha and Pichia pastoris.
We have studied the in vitro inactivation/dissociation and subsequent reactivation/re-assembly of peroxisomal alcohol oxidases (AO) from the yeasts Hansenula polymorpha and Pichia pastoris. Both proteins are homo-oligomers consisting of eight identical subunits, each containing one FAD as the prosthetic group. They were both rapidly inactivated upon incubation in 80% glycerol, due to their dissociation into the constituting subunits, which however still contained FAD. Dilution of dissociated AO in neutral buffer lead to reactivation of the protein due to AO re-assembly, as was demonstrated by non-denaturing PAGE. After use of mixtures of purified AO from H. polymorpha and P. pastoris active hybrid AO oligomers were formed. When prior to dissociation FAD was chemically removed from AO, reactivation or re-assembly did not occur independent of externally added FAD.
Evers
M E
ME
Department of Microbiology, University of Groningen, Haren, The Netherlands.
Harder
W
W
Veenhuis
M
M
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008830:Microbodies / Q000201:enzymology; D010843:Pichia / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation
D005182:Flavin-Adenine Dinucleotide; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase