Lys515-Lys492 cross-linking by DIDS interferes with substrate utilization by the sarcoplasmic reticulum ATPase.
Sarcoplasmic reticulum (SR) Ca2+ ATPase was derivatized with 4,4'-diisothiocyanatostilbene-2,2'-sulfonic acid (DIDS), and complete enzyme inactivation was produced with a molecular stoichiometry of one DIDS per ATPase. It was determined by peptide analysis and sequencing that Lys492 and Lys515 were the ATPase residues derivatized by DIDS. Lack of electrophoretic resolution of the two peptide fragments that result from a single tryptic cut at Arg505 demonstrated that the two derivatized residues were cross-linked. Cross-linking of Lys492 and Lys515 by DIDS interfered with ATPase utilization of both ATP and p-nitrophenyl phosphate substrates, whereas derivatization of only Lys515 with fluorescein isothiocyanate interfered with ATPase utilization of ATP but not of p-nitrophenyl phosphate. Cross-linking with DIDS implies a distance of approximately 13 A between Lys492 and Lys515, which corresponds to the length of ATP bound in an extended configuration. Therefore, within the groove of the nucleotide binding domain, the ATP substrate is positioned with the adenosine moiety near Lys515 and its terminal phosphate near Lys492.
Hua
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S
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore 21201, USA.
Inesi
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008239:Lysine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid