title: In vivo inactivation of peroxisomal alcohol oxidase in Hansenula polymorpha by KCN is an irreversible process.
abstract: The fate of alcohol oxidase (AO) in chemostat-grown cells of Hansenula polymorpha, after its inactivation by KCN, was studied during subsequent cultivation of the cyanide-treated cells in fresh methanol media. Biochemical experiments showed that the cyanide-induced inactivation of AO was due to the release of flavin adenine dinucleotide (FAD) from the holo enzyme. However, dissociation of octameric AO into subunits was not observed. Subsequent growth of intact cyanide-treated cells in fresh methanol media was paralleled by proteolytic degradation of part of the peroxisomes present in the cells. The recovery of AO activity, concurrently observed in these cultures, was accounted for by synthesis of new enzyme protein. Reactivation of previously inactivated AO was not observed, even in the presence of FAD in such cultures. Newly synthesized AO protein was incorporated in only few of the peroxisomes present in the cells. 31P nuclear magnetic resonance (NMR) studies showed that cyanide-treatment of the cells led to a dissipation of the pH gradient across the peroxisomes membrane. However, restoration of this pH gradient was fast when cells were incubated in fresh methanol medium after removal of the cyanide.
authors.affiliation: Department of Microbiology, University of Groningen, Haren, The Netherlands.
authors.forename: I J
authors.initials: IJ
authors.lastname: van der Klei
authors.forename: M
authors.initials: M
authors.lastname: Veenhuis
authors.forename: K
authors.initials: K
authors.lastname: Nicolay
authors.forename: W
authors.initials: W
authors.lastname: Harder
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors*; D003486:Cyanides / Q000494:pharmacology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D008830:Microbodies / Q000201:enzymology; D008854:Microscopy, Electron; D010843:Pichia / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D011190:Potassium Cyanide / Q000494:pharmacology*; D004718:Saccharomycetales / Q000378:metabolism*
chemical_list: D003486:Cyanides; D005656:Fungal Proteins; D005182:Flavin-Adenine Dinucleotide; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D011190:Potassium Cyanide