IN-VITRO DISSOCIATION AND RE-ASSEMBLY OF PEROXISOMAL ALCOHOL OXIDASESOF 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 ho mo-oligomers consisting of eight identical subunits, each containing o ne FAD as the prosthetic group. They were both rapidly inactivated upo n incubation in 80% glycerol, due to their dissociation into the const ituting subunits, which however still contained FAD. Dilution of disso ciated 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 mere formed, When prior to dissociation FAD was ch emically removed from AO, reactivation or re-assembly did not occur in dependent of externally added FAD.