In vivo inactivation of peroxisomal alcohol oxidase in Hansenula polymorpha by KCN is an irreversible process |
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Authors: | Ida J van der Klei Marten Veenhuis Klaas Nicolay Wim Harder |
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Institution: | (1) Department of Microbiology, Biological Centre, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands;(2) Laboratory for Electron Microscopy, Biological Centre, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands;(3) Institute of Molecular Biology and Medical Biotechnology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands |
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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.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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Keywords: | Hansenula polymorpha Peroxisomes Alcohol oxidase Protein assemblage FAD Cyanide |
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