As part of a long-term study of the spatial relationships of the proteins of the mammalian inner mitochondrial membrane, with emphasis upon the polypeptides of the electron transport chain, the applicant proposes to investigate the molecular morphology and topography of bovine heart mitochondrial NADH-ubiquinone reductase in a three phase project. In the first phase, the specific aim is to examine the molecular morphology of the soluble form of NADH-ubiquinone reductase (complex I) and determine the effects of substrates and inhibitors such as rotenone, Demerol, and barbituates such as amytal and Seconal on morphology. In the second phase the aims are to determine the morphology of a reconstituted enzyme preparation as in the first phase and to compare it to the soluble form of complex I.
The aims of the third phase are to examine the morphology of the native membrane bound enzyme and compare it to the other two forms of the protein. To accomplish these aims, the applicant proposes to (a) chemically crosslink the three forms of the enzyme with a variety of cleavable bifunctional reagents, (b) solubilize the crosslinked the membrane bound form of the enzyme with detergent (phase three), (c) isolate the crosslinked products by chromatography on immobilized antibody to complex I (phase three), and (d) separate the products by two dimensional polyacrylamide gel electrophoresis (with sodium dodecyl sulfate and a 2-20% gradient gel in both dimensions). The crosslinker will be cleaved after the first dimension. Analysis of control and crosslinked electrophoretograms in phase one and two will be by visual inspection of stained gels, and in phase three by transfer to nitrocellose and Western blotting involving and overlay of anti-complex I antibody or antibody to subfractions of the enzyme, then overlay with anti-rabbit-IgG conjugated to horseradish peroxidase, followed by enzymatic color development using 1-chloro-4-naphthol. By using crosslinkers of different bridging distances and membrane solubility, it should be possible to develop a three dimensional picture of the molecular morphology and dusbunit interactions of this enzyme in a detail not possible before. This project may also provide clues about the mechanism of malfunction of the enzyme reported to occur during anemia, anoxia, cystic fibrosis, and certain mitochondrial myopathies.
| Anderson, W M; Delinck, D L; Benninger, L et al. (1993) Cytotoxic effect of thiacarbocyanine dyes on human colon carcinoma cells and inhibition of bovine heart mitochondrial NADH-ubiquinone reductase activity via a rotenone-type mechanism by two of the dyes. Biochem Pharmacol 45:691-6 |
| Anderson, W M; Chambers, B B; Wood, J M et al. (1991) Inhibitory effects of two structurally related carbocyanine laser dyes on the activity of bovine heart mitochondrial and Paracoccus denitrificans NADH-ubiquinone reductase. Evidence for a rotenone-type mechanism. Biochem Pharmacol 41:677-84 |
| Anderson, W M; Patheja, H S; Delinck, D L et al. (1989) Inhibition of bovine heart mitochondrial and Paracoccus denitrificans NADH----ubiquinone reductase by dequalinium chloride and three structurally related quinolinium compounds. Biochem Int 19:673-85 |
