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Chemical cross-linking of mitochondrial NADH dehydrogenase from bovine heart.

ABSTRACT: The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by using two cleavable cross-linking agents, disuccinimidyl tartrate and (ethylene glycol)yl bis-(succinimidyl succinate). Cross-linking was analysed primarily by immunoblotting to detect products containing subunits of the iron-protein fraction from chaotropic resolution of the enzyme, namely those of 75, 49, 30 and 13 kDa. By using both the isolated iron-protein fraction and the intact dehydrogenase, cross-links were identified between these four subunits, from these subunits to the largest subunit of the flavoprotein fraction, which contains the active site for NADH, and from these subunits to polypeptides in the hydrophobic shell, which surrounds the hydrophilic iron-protein and flavoprotein fractions.

SUBMITTER: Cleeter MW

PROVIDER: S-EPMC1144865 | biostudies-other | 1985 Apr

REPOSITORIES: biostudies-other

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Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking.

Project description:The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by cross-linking constituent subunits with disuccinimidyl tartrate, (ethylene glycol)yl bis(succinimidyl succinate) and dimethyl suberimidate. Cross-linked products were identified by Western blotting with monospecific antisera to nine subunits of the enzyme. Cross-links between subunits within the flavoprotein, iron-protein and hydrophobic domains of the enzyme were identified. Cross-linking between the 75 kDa iron-protein-domain subunit and the 51 kDa flavoprotein-domain subunit was modulated by the substrate NADH. Cross-linking of subunits of the iron-protein and flavoprotein domains to constituents of the hydrophobic domain was also found. This was further substantiated by photolabelling subunits of the latter region, which were in contact with the membrane lipid, with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. One such subunit of Mr 19,000 could be cross-linked to components of the iron-protein domain.

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