Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase. Proteolytic and thermal inactivation of the membrane component.
Pyridine dinucleotide transhydrogenase of the Rhodospirillum rubrum chromatophore membrane was readily resolved by a washing procedure into two inactive components, a soluble transhydrogenase factor protein and an insoluble membrane-bound factor. Transhydrogenation was reconstituted on reassociation of these components. The capacity of the membrane factor to reconstitute enzymatic activity was lost after proteolysis of soluble transhydrogenase factor-depleted membranes with trypsin. NADP+ or NADPH, but neither NAD+ nor NADH, stimulated by several fold the rate of trypsin-dependent inactivation of the membrane factor. Substantial protection of the membrane factor from proteolytic inactivation was observed in the presence of Mg2+ ions, an inhibitor of transhydrogenation, or when the soluble transhydrogenase factor was bound to the membrane. Coincident with the loss of enzymatic reconstitutive capacity of the membrane factor was a loss in the ability of the membranes to bind the soluble transhydrogenase factor in a stable complex. The membrane component was inactivated by preincubating soluble transhydrogenase factor-depleted membranes at temperatures above 45 degrees. NADP+, NADPH, or Mg2+ ions, but neither NAD+ nor NADH, protected against inactivation. These studies indicate that (a) the binding of NADP+ or NADPH to the membrane factor promotes a conformational alteration in the protein such that its themostability and susceptibility to proteolysis are increased, and (b) the inhibitory Mg2+ ion-binding site resides in the membrane component.
D020130:Bacterial Chromatophores / Q000201:enzymology; D001665:Binding Sites; D004355:Drug Stability; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008566:Membranes / Q000201:enzymology; D009243:NAD; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009249:NADP; D010084:Oxidation-Reduction; D011485:Protein Binding; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors; D014357:Trypsin
D046911:Macromolecular Substances; D009243:NAD; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D014357:Trypsin; D008274:Magnesium