Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria

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Abstract
The external NADH dehydrogenase was purified from A. maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulfate/polyacrylamide-gel electrophoresis shows, that when the gel is silver-stained, the purified enzyme contains 2 major bands of MW 78,000 and 65,000 and a minor 1 of MW .apprx. 76,000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly Fe-S centers, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme was distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.