Studies on reconstitution of the Rhodospirillum rubrum nicotinamide nucleotide transhydrogenase.
The energy-transducing nicotinamide nucleotide transhydrogenase of Rhodospirillum rubrum is composed of 3 subunits alpha 1, alpha 2 and beta, with M(r) values, respectively, of 40.3, 14.9 and 47.8 kDa. Subunit alpha 1 is water-soluble, loosely bound to chromatophores, and can be easily and reversibly removed. Subunits alpha 2 and beta are integral membrane proteins, and their removal from chromatophores requires the use of detergents. Treatment of chromatophores with various detergents inhibited reconstitution of transhydrogenase activity when alpha 1 was added to the detergent-treated chromatophores. This apparent inhibition could be reversed by addition of a divalent metal ion. The best condition for extraction of alpha 2/beta from chromatophores was the use of 1% deoxycholate in the presence of 0.34 M KCl. Under these conditions, the extracted alpha 2/beta mixed with purified alpha 1 was completely inactive, but gained full activity when the assay medium was supplemented with 2-3 mM MgCl2 or CaCl2. It was shown that metal ions had little effect on the apparent Km of substrates, but greatly increased the affinity between purified alpha 1 and the detergent-treated or detergent-solubilized alpha 2/beta. It seems possible that the R. rubrum transhydrogenase contains a detergent-extractable metal ion, which is required for proper binding of the soluble alpha 1 subunit to the chromatophore-bound alpha 2/beta subunits.
Milgrom
Y M
YM
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037, USA.
Hatefi
Y
Y
D020130:Bacterial Chromatophores / Q000187:drug effects / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride / Q000494:pharmacology; D009250:NADP Transhydrogenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012995:Solubility
D003902:Detergents; D015636:Magnesium Chloride; D009250:NADP Transhydrogenases; D008274:Magnesium; D002118:Calcium