Abstract
The cysteinyl peptides of the gamma subunit of chloroplast coupling factor 1 (CF1) have been analyzed by high performance liquid chromatography. Analysis of the reduced enzyme alkylated with 4-vinylpyridine showed that the gamma subunit contains four cysteinyl residues. Two of these residues are involved in a disulfide linkage in CF1 either in solution or bound to washed thylakoid membranes. Two free sulfhydryls, one that is readily attacked by alkylating reagents and another that is less reactive, were also detected. Each of these four cysteinyl residues is present in a separate tryptic peptide derived from the gamma subunit. These results show that 4-vinylpyridine is an excellent reagent for the analysis of cysteinyl-containing peptides and support our analyses of the roles of cysteinyl residues in the gamma subunit in ATP synthesis and hydrolysis.
Highlights
EXPERIMENTALPROCEDURES linkage in CF, either in solution or bound to washed thylakoid membranes
For N-ethylmaleimide modification of membrane-bound CF, thylakoids equivalent to 0.15 mg/ml chlorophyll were incubated in 50 mM Tricine-NaOH(pH 8.0), 50 mM NaC1, 5 mM MgCl, 20 p M pyocyanine, and 2 mM [3H]N-ethylmaleimide in either white light
For that 4-vinylpyridineisanexcellent reagent for the CF, labeled exclusively at the“light” site with [3H]N-ethylmaleimide, analysis of cysteinyl-containing peptides and support thylakoids were first incubated in the dark for 2 min with 2 mM N
Summary
0.1% trifluoroacetic acid (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B). In acid (0.1% phosphoric acid), PEC has an extinction coefficient a t 252 nm of 4.75 mM" cm", more than 10 times that of eitherphenylalanine or tyrosine This high absorbance allows the cysteine-containing peptides modified with vinylpyridine to be identified during HPLC runs and to be collected for further analysis. The dark-labeled peptide,which corresponds to of each was determined These analyses showed that there the S4 sulfhydryl, elutes 8 min after the major peak of the were four different PEC-containing peptides labeled S1, S2, light-labeledsample. This is most consistent with the site. BThisis the total of a PEC determination and cysteic acid determination
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