Cysteine 254 of the 73-kDa A subunit is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by sulfhydryl reagents.
The vacuolar class of (H+)-ATPases are highly sensitive to sulfhydryl reagents, such as N-ethylmaleimide. The cysteine residue which is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by N-ethylmalemide is located in subunit A and is able to form a disulfide bond with the cysteine moiety of cystine through an exchange reaction. This unique property distinguishes this cysteine residue from the remaining cysteine residues of the (H+)-ATPase. Using this reaction, we selectively labeled the cystine-reactive cysteine residue of subunit A with fluorescein-maleimide. After complete digestion of the labeled subunit A by V8 protease, a single labeled fragment of molecular mass 3.9 kDa was isolated and the amino-terminal sequence was determined. This fragment contains 2 cysteine residues, Cys240 and Cys254. Since Cys254 is conserved among all vacuolar (H+)-ATPases whereas Cys240 is not, it is likely that Cys254 is the residue which is responsible for the sensitivity of the vacuolar (H+)-ATPase to sulfhydryl reagents.
Feng
Y
Y
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Forgac
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002966:Clathrin / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D003545:Cysteine; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D002966:Clathrin; D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol