Archives of Microbiology

, Volume 151, Issue 1, pp 26–33 | Cite as

In vivo inactivation of peroxisomal alcohol oxidase in Hansenula polymorpha by KCN is an irreversible process

  • Ida J. van der Klei
  • Marten Veenhuis
  • Klaas Nicolay
  • Wim Harder
Original Papers

Abstract

The fate of alcohol oxidase (AO) in chemostatgrown 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 paralelled 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 peroxisomal membrane. However, restoration of this pH gradient was fast when cells were incubated in fresh methanol medium after removal of the cyanide.

Key words

Hansenula polymorpha Peroxisomes Alcohol oxidase Protein assemblage FAD Cyanide 

Abbreviations

AO

alcohol oxidase

FAD

flavin adenine dinucleotide

CHI

cycloheximide

NMR

nuclear magnetic resonance

FPLC

fast protein liquid chromatography

RIE

rocket immuno electrophoresis

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Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Ida J. van der Klei
    • 1
  • Marten Veenhuis
    • 2
  • Klaas Nicolay
    • 3
  • Wim Harder
    • 1
  1. 1.Department of Microbiology, Biological CentreUniversity of GroningenHarenThe Netherlands
  2. 2.Laboratory for Electron Microscopy, Biological CentreUniversity of GroningenHarenThe Netherlands
  3. 3.Institute of Molecular Biology and Medical BiotechnologyUniversity of UtrechtUtrechtThe Netherlands

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