Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

• Published in: European journal of biochemistry Vol. 111; no. 2; pp. 347 - 355
• Main Authors: VOORDOUW, Gerrit, VIES, Saskia M., EWEG, Jan Kees, VEEGFR, Cees, VAN BREEMEN, Jan F. L., VAN BRUGGEN, Ernst F. J.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.10.1980
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1980.tb04948.x

1 Pyridine nucleotide transhydrogenase from Azotobacter vinelandii was purified with a scaled‐up procedure. In a typical purification 500 ml cell‐free extract from 200 g cells is loaded on an Ado‐2′, 5′‐P2‐Sepharose 4B affinity column (20 ml bed volume). After washing, the enzyme is desorbed with 2′AMP at neutral pH and further purified by Sephadex G‐200 gel chromatography. The enzyme (10–12 mg) is obtained in 40–60% yield and is homogeneous its judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. 2 The homogeneity of the purified enzyme is also apparent from electron microscopy studies, where the enzyme appears as a polydisperse set of polymers without contaminating, structures and from fluorescence lifetime studies by the method of single‐photon counting. The flavin fluorescence appears to decay with a single lifetime τ= 2.5 ns. The polymeric nature of transhydrogenase can be aptly demonstrated by density gradient centrifugation in the presence of KBr. After centrifuging for 50 h at 160000 × g and 10° C the enzyme is concentrated in a narrow fluorescent band with buoyant density Qb= 1.305 g cm−3. 3 The arrangement of subunits in the transhydrogenase polymer has been derived from optical diffraction studies of electron micrographs. The polymers are built tip from a linear assembly of tetramers. Four subunits are placed in a rhomb with sides of 13.5 mm and an angle of 45° (135°) between the sides. A second tetramer is located staggered on top of the first one. Since a variety of other studies have indicated that the polymers dissociate into octamers under alkaline condition [Voordouw, G. et al. (1979) Eur. J. Biochem. 98, 447–454] we conclude that this smallest functional unit is built up from two tetramers.