Studies on the interaction of bovine heart mitochondrial NADH dehydrogenase with phospholipids and inhibitors

Earley, Fergus Gerard Paul (1985) Studies on the interaction of bovine heart mitochondrial NADH dehydrogenase with phospholipids and inhibitors. University of Southampton, Doctoral Thesis.

Abstract

The ability of several rotenone derivatives and other rotenoid compounds to inhibit the mitochondrial NADH-ubiquinone oxidoreductase (EC 1.6.99.3) in the form of the purified lipoprotein complex from beef heart (Complex 1) was tested. The results were consistent with those of a previous study on intact mitochondrial membranes (Burgos and Redfearn, 1965). One of the active rotenoids, amorphigenin (8' hydroxy rotenone) was used as a nucleus for the synthesis of a radioactive, photoreactive, labelling reagent (N-(4-azido-2-nitrophenyl)-β-alanyl amorphigenin) which was as effective an inhibitor as rotenone. Irradiation of this compound in the presence of Complex 1, at wavelengths above 300 nm, caused no loss of inhibitory activity and resulted in low yield but specific incorporation of the label into a protein of apparent mol. wt. 33,000. The amount of this polypeptide present in two different Complex 1 preparations correlated with their sensitivity to inhibition by rotenone. This protein was shown to be a true constituent of Complex 1 by its presence in immunoprecipates. The hydrophobic labelling reagent (iodonapth-1-yl azide) and the photoreactive phospholipid analogues (1-acyl-2-[12-amino-N-(4-azido-2-nitrophenyl)dodecanoyl]-sn-glycero-3-phosphocholine and 1-palmitoyl-2-[4-nitro-2-azido]benzoyl-sn-glycero-3-phosphocholine) were used to identify those polypeptides of Complex 1 which were exposed to the lipid phase under different conditions. No labelling of the flavoprotein or iron protein subunits could be detected unless the complex was denatured by lipid depletion. Labelling was mostly restricted to polypeptides having apparent mol. wts. 39,000, 33,000, 20,000, 16,000, 15,000, 13,000, 12,000 and 5,000; these and other labelled polypeptides form part of the hydrophobic `P' fraction. Some evidence on the degree of exposure of certain polypeptides at different depths in the membrane, based on relative labelling by the two phospholipid probes, is also presented. The interaction of Complex 1 with phosphatidylcholine and cardiolipin was examined by the used of brominated phospholipids to quench the protein fluorescence. The experiments confirmed the existence of cardiolipin specific binding sites and revealed that the protein had a slight preference for cardiolipin at other phospholipid binding binding sites (K'_CL/DOL= 1.44). It was shown that cardiolipin alone can support activity. The synthesis of a photoreactive (but not radioactive) analogue of cardiolipin is reported. (D66111/86)

This record has no associated files available for download.

Contributors

Download statistics