Abstract
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.
Highlights
Themitochondrialproton-translocatingnicotinahydrophilic N-terminaldomain, a400-residue-longhydrophomide nucleotide transhydrogenase is embedded in the bic central domain, and a 200-residue-long hydrophilic C
Hydropathy analyses have suggested that the central hydrophobic domain is made up mainly of about 14 hydrophobic clusters of -20 residues each [4]. These hydrophobic clustersappear to intercalate into the mitochondrial inner membrane, and there appears to be little transhydrogenase mass protruding outward into the intermembrane space, exwas determined to be Alaeso-Alaesl, which is located in a small loop of the transhy- pairs of membrane-intercalating hydrophobic clusters [5]
The inactivation ratoef the bisected enzyme membrane) are treated with proteinase K, subtilisin, therby trypsin and N-ethylmaleimide was altered in the molysin, or pronase E,the transhydrogenase molecule is presence of NADP and NADPH, suggesting substrate- bisected into a 72-kDa N-terminal fragment and a 37-kDa C
Summary
The inactivation ratoef the bisected enzyme membrane) are treated with proteinase K, subtilisin, therby trypsin and N-ethylmaleimide was altered in the molysin, or pronase E,the transhydrogenase molecule is presence of NADP and NADPH, suggesting substrate- bisected into a 72-kDa N-terminal fragment and a 37-kDa C-. Shown to be Ala690-Ala69w1,hich is located within the large, like the intact enzyme, proteoliposomes of the cytosolic side-exposed loop mentioned above (see Fig. 1).The bisected transhydrogenase were capableof membrane proteinase K-nickedenzyme can be isolated and purified, and potential formation and internal acidificationcoupled this paper describes its enzymatic and proton-translocating to NADPH 4NAD transhydrogenation. The mitochondrial proton-translocating nicotinamide nucleotide transhydrogenase is a homodimer of monomer M , = 109,228 It is located in the mitochondrial inner membrane and catalyzes the reaction shown, where Hc+.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
