Abstract
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.
Highlights
Cysteine 254 of the 73-kDa A Subunit Is Responsible for Inhibition of the Coated Vesicle (H+)-ATPaseUpon Modification by Sulfhydryl Reagents*
The vacuolarclass of (H+)-ATPases arehighly sen- are inhibited by higher concentrations of NEM (0.1-1 mM), sitive to sulfhydryl reagents, suchas N-ethylmaleim- andthe F-type ATPases, which are virtually resistant to ide
When treated with NEM, the ATPase added to thevesicles to neutralize unreacted NEM and tocleave activity of this enzyme is irreversibly abolished. This enzyme disulfide bonds formed between cysteine residues of the protein and the cysteine moiety of cystine
Summary
The vacuolar class of (H+)-ATPases' carry out acidification Materials-Calf brains were obtained from a local slaughterhouse. Preparation of Clathrin-coated Vesicle ATPase Modified by Cystine-Clathrin-coated vesicles were prepared from calf brain as previously described [2].Vesicleswere stripped of their clathrin coat according to Adachi et at. [19].Stripped vesicles weresuspended in 1 mM EDTA, 10% glycerol, and 20 mM HEPES (pH 7.0)at a protein concentration of 2 mg/ml. All vacuolar ATPases sharethe property of being inhibited by low concentrations of N-ethylmaleimide (NEM) The abbreviations used are: (H+)-ATPase,proton translocating protein) in 0.6 mlof 0.02% C,,Eg, 8 pgof phosphatidylcholine, 0.2 adenosine triphosphatase; NEM, N-ethylmaleimide; HEPES, 4-(2- mM EGTA, 10% glycerol, and 20 mM HEPES (pH7.0).The mixtures hydroxyethy1)-1-piperazineethanesulfonicacid; EGTA, [ethylene- were incubated for 10 min a t 23 "C. Labeling of the Coated Vesicle (H+)-ATPase with Fluorescein Maleimide-Stripped vesicles wereincubated with cystine until no proton transport could be detected as described above. NEM (5 mM) was added to thecystine-treated stripped vesicles, and thereaction
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