In vitro dissociation and re‐assembly of peroxisomal alcohol oxidases of Hansenula polymorpha and Pichia pastoris

Abstract

We have studied the in vitro inactivation/dissociation and subsequent reactivation/re-assembly of peroxisomal alcohol oxidases (AO) from the yeasts Hansenulapolymorpha 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 snbunits, 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.

Routing ofHansenula polymorphaAlcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery

“…Purified native octameric AO was dissociated into monomers by incubation in 80% glycerol (final AO protein concentration 0.1 M) for 20 min at 23°C (Evers et al, 1995). To study interactions of HpPex5p with WT AO or the C-terminally truncated form AO⌬16, AO protein incubated in 80% glycerol was diluted 10 times in 50 mM potassium phosphate buffer, pH 6.0, containing 0.3 M purified HpPex5p (Evers et al, 1995).…”

“…To study interactions of HpPex5p with WT AO or the C-terminally truncated form AO⌬16, AO protein incubated in 80% glycerol was diluted 10 times in 50 mM potassium phosphate buffer, pH 6.0, containing 0.3 M purified HpPex5p (Evers et al, 1995). The samples were kept on ice for 15 min before being subjected to nondenaturating gel electrophoresis using 4 -10% polyacrylamide gradient gels (Musgrove et al, 1987).…”

Deflavination of flavo-oxidases by nucleophilic reagents

“…All protein solutions were in 50 mM potassium phosphate bu¡er, pH 7.5. Dissociation of native AO octamers into monomers was achieved in 60% glycerol [14,16]. H. polymorpha and P. pastoris AO, GOX and DAOX were depleted of FAD upon incubation with 6 mM of either KCN, NaSCN or KCNO for 60^80 min at 10³C followed by dialysis for 24 h at 10³C against phosphate bu¡er, pH 7.5.…”

“…Therefore, we checked whether the e¡ects of the three nucleophilic reagents (KCN, NaSCN and KCNO) on AO protein were dependent on the oligomeric state or a¡ected the FAD-binding in monomers as well. For this purpose, P. pastoris AO was ¢rst dissociated into subunits by treatment with 60% glycerol [14,16], subsequently CN 3 , SCN 3 or CNO 3 ions were added and ¢nallŷ upon incubation for 60 min^dialyzed against glycerol-containing bu¡er in order to keep the protein in the monomeric conformation. The nucleophilic reagents caused dissociation of FAD and similar spectroscopic changes as those observed with the octamers (data not shown).…”

Octameric alcohol oxidase dissociates into stable, soluble monomers upon incubation with dimethylsulfoxide

“…Incubation of native AO in 80% glycerol resulted in dissociation of the octamers into the constituting subunits. However, FAD remained bound to these subunits [7,8]. Moreover, the presence of a highly viscous solvent overshadows the results of studies of the dynamic properties of proteins and is likely to exert unwanted side effects on HpPyc1p in reconstitution experiments.…”