Bovine factor B: cloning, expression, and characterization.
Bovine factor B, a polypeptide required for the coupled activity of the mitochondrial ATP synthase complex, was cloned. A novel expression system for overproducing the recombinant bovine factor B was developed, which yielded the recombinant polypeptide at a level of 12-15 mg of protein per liter of bacterial culture. Reconstitution of the recombinant polypeptide with factor B-depleted ammonia, EDTA-treated submitochondrial particles (AE-SMP) restored the formation of substrate-driven DeltapH gradient across vesicular membranes, presumably by blocking a proton leak. The proton leak in the AE-SMP could also be blocked by the F0 inhibitors oligomycin and dicyclohexylcarbodiimide, but not the F1-ATPase inhibitors efrapeptin and aurovertin B. The six factor B thiols titrated rapidly with Ellman's reagent, and two of these, presumably Cys92 and Cys94, gained protection following treatment of factor B with a vicinal dithiol-specific reagent phenylarsine oxide (PAO). Similarly, Cd2+, whose binding to factor B is believed to also involve a vicinal dithiol, and PAO, protected approximately 2 Cys residues against labeling with sulfhydryl-specific fluorescent reagent fluorescein-5'-maleimide. The circular dichroism spectra showed that binding of Cd2+ and Zn2+, but not Ca2+ to bovine factor B caused small but reproducible changes in the secondary structure elements of the polypeptide.
Belogrudov
Grigory I
GI
Department of Physiology, David Geffen School of Medicine, University of California at Los Angeles, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA. gbelo@ucla.edu
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003001:Cloning, Molecular; D011415:Complement Factor B / Q000235:genetics* / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014050:Toluene / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D001152:Arsenicals; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D002104:Cadmium; C029341:oxophenylarsine; D014050:Toluene; D011415:Complement Factor B; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D015032:Zinc; D003545:Cysteine; D002118:Calcium; C004848:dithiol