Abstract:
:Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Miller RP,Farley RAdoi
10.1021/bi00458a025subject
Has Abstractpub_date
1990-02-13 00:00:00pages
1524-32issue
6eissn
0006-2960issn
1520-4995journal_volume
29pub_type
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