The structure of mitochondrial NADH dehydrogenase
The structure of mitochondrial NADH dehydrogenase
Antisera to Complex I (NADH-ubiquinone oxidoreductase, EC 1.6.99.3.) purified from bovine heart mitochondria, and to fractions from this enzyme have been used to study the structure of mitochondrial NADH dehydrogenase. Analysis by one and two dimensional electrophoretic techniques resolved Complex I into 26 different polypeptides. Similar analyses of immunoprecipitates formed by allowing the antisera to react with solubilised Complex I or submitochondrial particles showed that these 26 polypeptides are true constituents of the enzyme. The NADH dehydrogenase content of mitochondria and submitochondrial particles was measured by (I) rocket immunoclectrophoresis to determine the protein content and (II) the fluorescence of the FM prosthetic group. The turnover number of Complex I and NADH dehydrogenase isolated from solubilised submitochondrial particles by immunoprecipitation were found to be very similar. The topography of NADH dehydrogenase polypeptides in the inner mitochondrial membrane was studied using the membrane impermeable probes, diazobenzene sulphate and lactoperoxidase catalysed iodination to label proteins on the outside of the membrane. Intact mitochondria and fully inverted submitochondrial particles were labelled, solubilised and allowed to react with antiserum. Analysis of labelled immunoprecipitates showed that NADH dehydrogenase was asymmetrically organised and spanned the inner mitochondrial membrane. Antisera to Complex I were shown to cross-react with the corresponding rat heart enzyme. Some evidence has been presented to suggest that biosynthesis of the whole enzyme in isolated rat heart slices involves products of both nucleocytoplasmic and mitochondrial protein synthesis. A model for the structure of mitochondrial NADH dehydrogenase has been suggested on the basis of these results.
University of Southampton
1981
Smith, Stuart Kevin
(1981)
The structure of mitochondrial NADH dehydrogenase.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Antisera to Complex I (NADH-ubiquinone oxidoreductase, EC 1.6.99.3.) purified from bovine heart mitochondria, and to fractions from this enzyme have been used to study the structure of mitochondrial NADH dehydrogenase. Analysis by one and two dimensional electrophoretic techniques resolved Complex I into 26 different polypeptides. Similar analyses of immunoprecipitates formed by allowing the antisera to react with solubilised Complex I or submitochondrial particles showed that these 26 polypeptides are true constituents of the enzyme. The NADH dehydrogenase content of mitochondria and submitochondrial particles was measured by (I) rocket immunoclectrophoresis to determine the protein content and (II) the fluorescence of the FM prosthetic group. The turnover number of Complex I and NADH dehydrogenase isolated from solubilised submitochondrial particles by immunoprecipitation were found to be very similar. The topography of NADH dehydrogenase polypeptides in the inner mitochondrial membrane was studied using the membrane impermeable probes, diazobenzene sulphate and lactoperoxidase catalysed iodination to label proteins on the outside of the membrane. Intact mitochondria and fully inverted submitochondrial particles were labelled, solubilised and allowed to react with antiserum. Analysis of labelled immunoprecipitates showed that NADH dehydrogenase was asymmetrically organised and spanned the inner mitochondrial membrane. Antisera to Complex I were shown to cross-react with the corresponding rat heart enzyme. Some evidence has been presented to suggest that biosynthesis of the whole enzyme in isolated rat heart slices involves products of both nucleocytoplasmic and mitochondrial protein synthesis. A model for the structure of mitochondrial NADH dehydrogenase has been suggested on the basis of these results.
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Published date: 1981
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Local EPrints ID: 459555
URI: http://eprints.soton.ac.uk/id/eprint/459555
PURE UUID: 957f5d5f-45bd-4a06-a038-0b930171a81c
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Date deposited: 04 Jul 2022 17:14
Last modified: 04 Jul 2022 17:14
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Author:
Stuart Kevin Smith
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