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.
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
Grigory I
GI
Belogrudov
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