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Purification, Characterization, and Submitochondrial Localization of the 32-Kilodalton NADH Dehydrogenase from Maize
A. F. Knudten1, J. J. Thelen, M. H. Luethy
1School of Biological Sciences and the Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0118.
Abstract:
Plant mitochondria have the unique ability to directly oxidize exogenous NAD(P)H. We recently separated two NAD(P)H dehydrogenase activities from maize (Zea mays L.) mitochondria using anion-exchange (Mono Q) chromatography. The first peak of activity oxidized only NADH, whereas the second oxidized both NADH and NADPH. In this paper we describe the purification of the first peak of activity to a 32-kD protein. Polyclonal antibodies to the 32-kD protein were used to show that it was present in mitochondria from several plant species. Two-dimensional gel analysis of the 32-kD NADH dehydrogenase indicated that it consisted of two major and one minor isoelectric forms. Immunoblot analysis of submitochondrial fractions indicated that the 32-kD protein was enriched in the soluble protein fraction after mitochondrial disruption and fractionation; however, some association with the membrane fraction was observed. The membrane-impermeable protein cross-linking agent 3,3[prime] -dithiobis-(sulfosuccinimidylpropionate) was used to further investigate the submitochondrial location of the 32-kD NADH dehydrogenase. The 32-kD protein was localized to the outer surface of the inner mitochondrial membrane or to the intermembrane space. The pH optimum for the enzyme was 7.0. The activity was found to be severely inhibited by p-chloromercuribenzoic acid, mersalyl, and dicumarol, and stimulated somewhat by flavin mononucleotide.
Insights
Researchers purified a 32-kD NADH dehydrogenase from maize mitochondria. This enzyme is located on the outer surface of the inner mitochondrial membrane or in the intermembrane space, suggesting a role in external NADH oxidation.
Area of Science:
- Mitochondrial biochemistry
- Plant physiology
- Enzyme purification
Background:
- Plant mitochondria can oxidize external NAD(P)H.
- Two distinct NAD(P)H dehydrogenase activities were previously identified in maize mitochondria.
- These activities were separated using anion-exchange chromatography.
Purpose of the Study:
- To purify the NADH-specific dehydrogenase activity.
- To characterize the purified protein and determine its submitochondrial localization.
- To investigate the enzyme's properties and distribution across plant species.
Main Methods:
- Anion-exchange chromatography (Mono Q) for enzyme separation.
- Protein purification to a 32-kD polypeptide.
- Antibody production and immunoblot analysis for species distribution.
- Two-dimensional gel electrophoresis for isoelectric forms.
- Submitochondrial fractionation and cross-linking studies for localization.
- Enzyme activity assays at varying pH and with inhibitors.
Main Results:
- A 32-kD protein responsible for NADH oxidation was purified.
- Antibodies confirmed the protein's presence in various plant species' mitochondria.
- The enzyme exists in multiple isoelectric forms.
- Localization studies indicated the protein is on the outer surface of the inner mitochondrial membrane or in the intermembrane space.
- The optimal pH is 7.0, with inhibition by specific agents and stimulation by flavin mononucleotide.
Conclusions:
- The purified 32-kD protein is a plant mitochondrial NADH dehydrogenase.
- Its location suggests a role in oxidizing external NADH.
- The enzyme is conserved across different plant species.