Cyclic reactions catalysed by detergent-dispersed and reconstituted transhydrogenase from beef heart mitochondria; implications for the mechanism of proton translocation
Sazanov LA, Jackson B. 1995. Cyclic reactions catalysed by detergent-dispersed and reconstituted transhydrogenase from beef heart mitochondria; implications for the mechanism of proton translocation. Biochimica et Biophysica Acta - Bioenergetics. 1231(3), 304–312.
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Journal Article
| Published
| English
Author
Sazanov, Leonid AISTA ;
Jackson, Baz
Abstract
Transhydrogenase from beef-heart mitochondria was solubilised with Triton X-100 and purified by column chromatography. The detergent-dispersed enzyme catalysed the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH, but only in the presence of NADP+. Experiments showed that this reaction was cyclic; NADP(H), whilst remaining bound to the enzyme, was alternately reduced by NADH and oxidised by AcPdAD+. A period of incubation of the enzyme with NADPH at pH 6.0 led to inhibition of the simple transhydrogenation reaction between AcPdAD+ and NADPH. However, after such treatment, transhydrogenase acquired the ability to catalyse the (NADPH-dependent) reduction of AcPdAD+ by NADH. It is suggested that this is a similar cycle to the one described above. Evidently, the binding affinity for NADP+ increases as a consequence of the inhibition process resulting from prolonged incubation with NADPH. The pH dependences of simple and cyclic transhydrogenation reactions are described. Though more complex than those in Escherichia coli transhydrogenase, they are consistent with the view [Hutton, M., Day, J.M., Bizouarn, T. and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041–10511] that, also in the mitochondrial enzyme, binding the release of NADP+ and NADP are accompanied by binding and release of a proton. The enzyme was successfully reconstituted into liposomes by a cholate dilution procedure. The proteoliposomes catalysed cyclic NADPH-dependent reduction of AcPdAD+ by NADH only when they were tightly coupled. However, they catalysed cyclic NADP+-dependent reduction of AcPdAD+ by NADH only when they were uncoupled eg. by addition of carbonylcyanide-p-trifluoromethoxyphenyl hydrazone. These observations are evidence that the proton binding and release which accompany NADP+ binding and release, respectively, take place on the inside of the vesicle, and that they are components of the electrogenic processes of the enzyme.
Publishing Year
Date Published
1995-10-10
Journal Title
Biochimica et Biophysica Acta - Bioenergetics
Publisher
Elsevier
Acknowledgement
L.A.S. is grateful to the Wellcome Trust for a Research Fellowship. Support from the Biotechnology and Biological Sciences Research Council is also acknowledged. We thank our colleagues. Tania Bizouarn, Mike Hutton and Nick Cotton, for advice and valuable discussion.
Volume
1231
Issue
3
Page
304 - 312
ISSN
IST-REx-ID
Cite this
Sazanov LA, Jackson B. Cyclic reactions catalysed by detergent-dispersed and reconstituted transhydrogenase from beef heart mitochondria; implications for the mechanism of proton translocation. Biochimica et Biophysica Acta - Bioenergetics. 1995;1231(3):304-312. doi:10.1016/0005-2728(95)00096-2
Sazanov, L. A., & Jackson, B. (1995). Cyclic reactions catalysed by detergent-dispersed and reconstituted transhydrogenase from beef heart mitochondria; implications for the mechanism of proton translocation. Biochimica et Biophysica Acta - Bioenergetics. Elsevier. https://doi.org/10.1016/0005-2728(95)00096-2
Sazanov, Leonid A, and Baz Jackson. “Cyclic Reactions Catalysed by Detergent-Dispersed and Reconstituted Transhydrogenase from Beef Heart Mitochondria; Implications for the Mechanism of Proton Translocation.” Biochimica et Biophysica Acta - Bioenergetics. Elsevier, 1995. https://doi.org/10.1016/0005-2728(95)00096-2.
L. A. Sazanov and B. Jackson, “Cyclic reactions catalysed by detergent-dispersed and reconstituted transhydrogenase from beef heart mitochondria; implications for the mechanism of proton translocation,” Biochimica et Biophysica Acta - Bioenergetics, vol. 1231, no. 3. Elsevier, pp. 304–312, 1995.
Sazanov LA, Jackson B. 1995. Cyclic reactions catalysed by detergent-dispersed and reconstituted transhydrogenase from beef heart mitochondria; implications for the mechanism of proton translocation. Biochimica et Biophysica Acta - Bioenergetics. 1231(3), 304–312.
Sazanov, Leonid A., and Baz Jackson. “Cyclic Reactions Catalysed by Detergent-Dispersed and Reconstituted Transhydrogenase from Beef Heart Mitochondria; Implications for the Mechanism of Proton Translocation.” Biochimica et Biophysica Acta - Bioenergetics, vol. 1231, no. 3, Elsevier, 1995, pp. 304–12, doi:10.1016/0005-2728(95)00096-2.
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