Mitochondrial energy-transducing nicotinamide nucleotide transhydrogenase. Purification and properties of the proteinase K-bisected enzyme.
The mitochondrial proton-translocating nicotinamide nucleotide transhydrogenase is embedded in the inner membrane as a homodimer of monomer Mr = 109,288. Its N-terminal 430 residues and C-terminal 200 residues protrude into the matrix, whereas its central 400 residues appear to intercalate into the inner membrane as 14 hydrophobic clusters of about 20 residues each (Yamaguchi, M., and Hatefi, Y. (1991) J. Biol. Chem. 266, 5728-5735). Treatment of mitoplasts (mitochondria denuded of outer membrane) with several proteolytic enzymes cleaves the transhydrogenase into a 72-kDa N-terminal and a 37-kDa C-terminal fragment. The cleavage site of proteinase K was determined to be Ala690-Ala691, which is located in a small loop of the transhydrogenase exposed on the cytosolic side of the inner membrane. This paper shows that the bisected transhydrogenase can be purified from proteinase K-treated mitoplasts with retention of greater than or equal to 85% transhydrogenase activity. The inactivation rate of the bisected enzyme by trypsin and N-ethylmaleimide was altered in the presence of NADP and NADPH, suggesting substrate-induced conformation changes similar to those reported previously for the intact transhydrogenase. Also, like the intact enzyme, proteoliposomes of the bisected transhydrogenase were capable of membrane potential formation and internal acidification coupled to NADPH----NAD transhydrogenation. The properties of the bisected transhydrogenase have been discussed in relation to those of the two-subunit Escherichia coli transhydrogenase, the bisected lac permease (via gene restriction), and the fragmented and reconstituted bacteriorhodopsin.
Yamaguchi
M
M
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
Hatefi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D004735:Energy Transfer; D007700:Kinetics; D008564:Membrane Potentials; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009250:NADP Transhydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D011522:Protons; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D014357:Trypsin
D011522:Protons; D009250:NADP Transhydrogenases; D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K