Electron-microscopic studies on location of SH-groups in mitochondrial F1-ATPase using a ferritin label.
A new approach has been suggested for electron-microscopic study of the structure of mitochondrial F1-ATPase based on ferritin labeling. By means of sequential treatment with 2-iminothiolane and Nbs2 we obtained a modified ferritin (NbsSPrCNH-Ft) able to react with SH-groups of proteins and to form conjugates in which the protein and ferritin are bound by disulfide bonds. An electron-microscopic investigation of the negatively stained preparations of mitochondrial F1-ATPase, preincubated with modified ferritin, revealed such enzyme-ferritin conjugates. In case of modified ferritin, containing 360 mol SH-groups per mol protein, and F1-ATPase, pretreated with N-ethylmaleimide and then with dithiothreitol, conjugates were obtained in which ferritin molecules are bound to several (as many as four) of the six protein masses, comprising a bilayer molecule of the enzyme. Taking into consideration the biochemical data on the location of accessible SH-groups (only in alpha, gamma or epsilon subunits), it is inferred from the results obtained that one of the protein masses is a complex between beta subunit and at least one of the minor subunits located partially on the molecule's external side. This indicates the nonequivalence of different copies of the major subunits. Averaged images of the particles of the F1-F0 complex from bovine heart mitochondria and bacteria Micrococcus lysodeicticus were obtained. It was found that F0 component is bound to two adjacent protein masses of the F1-ATPase molecule. It is suggested that this binding may be due the nonequivalency of single-type major subunits.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004228:Dithionitrobenzoic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005293:Ferritins / Q000031:analogs & derivatives / Q000378:metabolism*; D008837:Micrococcus / Q000201:enzymology; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000201:enzymology; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology; D013438:Sulfhydryl Compounds / Q000032:analysis
D013438:Sulfhydryl Compounds; D005293:Ferritins; D004228:Dithionitrobenzoic Acid; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide; D004229:Dithiothreitol