Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

Abstract

1 Purified transhydrogenase from Azotobacter vinelandii forms very stable polymers at neutral pH. These show little tendency to dissociate upon dilution but can be disoociated by raising the pH. 2 The stability of transhydrogenase polymers towards dilution can be demonstrated with enzyme that is in part covalently bound to Sepharose. Non-covalently bound subunits are kept bound upon elution at pH 7.5 and can only be released by raising the pH of the eluting buffer (pH 9.0). The remaining dissociated immobilized polymers can be reconstituted by adding free transhydrogenase at a lower pH (7.5–8.5). Reconstitution of dissociated immobilized A. vinelandii polymers can also be achieved with enzyme from Pseudomonas aeruginosa, which demonstrates the close structural relationship between these two enzymes. 3 Differential centrifugation of cell suspensions lysed with the osmotic shock method in the presence of EDTA shows transhydrogenase to be present largely as the non-polymerized octamer (S20,w= 15 S). However, the distribution of the enzyme activity changes markedly when divalent cations (MgCl2, CaCl2) are included in the lysis buffer: up to 65% of the transhydrogenase activity then co-sediments with membranes and nucleic acids (30 min, 20000 × g). Nuclease treatment leads to disappearance of the rapidly sedimenting transhydrogenase fraction. These results are incompatible with a reversible divalent-cation-mediated self-association of the enzyme in vivo. We conclude, on the contrary, that the transhydrogenase octamer is partly immobilized in vivo by divalent-cation-mediated interactions with cell constituents (e.g. nucleic acids) preventing its self-association. 4 The reduction of NAD+ by NADPH catalyzed by cell-free extract transhydrogenase is not influenced by phosphate ions, while the reverse reaction is completely inhibited by 5 mM phosphate. This inhibition is released by adding excess divalent cations (10 mM CaCl2 or MgCl2). Since other metabolites (adenosine, 5′AMP, ADP, ATP) inhibit both activities more or less to the same extent it follows that the antagonistic action of phosphate and divalent cations is of primary importance in regulating the direction of intracellular transhydrogenation in A. vinelandii. 5 The existence of binding sites for these ions on the transhydrogenase subunit is indicated by studies on the inhibition of cell-free extract transhydrogenase with the divalent thiol reagent p-aminophenylarsenoxide. The rate of reaction is slowed down by magnesium and accelerated by phosphate ions.