Sarcoplasmic reticulum Ca(2+)-ATPase is an ion pump whose catalytic cycle includes the transient formation of an acyl phosphate at Asp(351), and fluorescein isothiocyanate is a covalent inhibitor of ATP binding to this pump, known to specifically derivatize Lys(515) in the nucleotide-binding site. It was previously found that an unusually stable, phosphorylated form of fluorescein-ATPase, with low fluorescence, is obtained following Ca (2+) loading with acetyl phosphate as energy source and then chelation with EGTA of Ca(2+) on the cytosolic side. Here we show that the phospho-linkage in this low fluorescent species is stable at alkaline pH, unlike the acyl phosphate at Asp(351). Moreover, the low fluorescence and stable phosphoryl group track together in primary and secondary tryptic subfragments, separated by SDS-PAGE after denaturation. Finally, normal fluorescence and absorbance are recovered upon treatment with alkaline phosphatase after extensive trypsinolysis. We conclude that the low fluorescent species is the result of the phosphoryl group being transferred from Asp (351) to the fluorescein moiety during pump reversal, yielding fluorescein monophosphate tethered to Ca(2+)-ATPase.
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Unexpected phosphoryl transfer from Asp351 to fluorescein attached to Lys515 in sarcoplasmic reticulum Ca2+-ATPase.
Biochemistry
June 2008
Institute of Infectious Diseases and Molecular Medicine, Division of Chemical Pathology, and National Health Laboratory Services, University of Cape Town, South Africa.
Sarcoplasmic reticulum Ca(2+)-ATPase is an ion pump whose catalytic cycle includes the transient formation of an acyl phosphate at Asp(351), and fluorescein isothiocyanate is a covalent inhibitor of ATP binding to this pump, known to specifically derivatize Lys(515) in the nucleotide-binding site. It was previously found that an unusually stable, phosphorylated form of fluorescein-ATPase, with low fluorescence, is obtained following Ca (2+) loading with acetyl phosphate as energy source and then chelation with EGTA of Ca(2+) on the cytosolic side. Here we show that the phospho-linkage in this low fluorescent species is stable at alkaline pH, unlike the acyl phosphate at Asp(351).
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Biochemistry
April 2004
Cell Biology Group, Biological Sciences Division, Fundamental Science Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
High-resolution crystal structures obtained in two conformations of the Ca-ATPase suggest that a large-scale rigid-body domain reorientation of approximately 50 degrees involving the nucleotide-binding (N) domain is required to permit the transfer of the gamma-phosphoryl group of ATP to Asp(351) in the phosphorylation (P) domain during coupled calcium transport. However, variability observed in the orientations of the N domain relative to the P domain in the different crystal structures of the Ca-ATPase following calcium activation and the structures of other P-type ATPases suggests the presence of conformational heterogeneity in solution, which may be modulated by contact interactions within the crystal. Therefore, to address the extent of conformational heterogeneity between these domains in solution, we have used fluorescence resonance energy transfer to measure the spatial separation and conformational heterogeneity between donor (i.
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June 1999
Biochemistry and Biophysics Section, Department of Molecular Biosciences, University of Kansas, Lawrence 66045-2106, USA.
Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, and functions to modulate rate-limiting conformational transitions involving the transport activity of the Ca-ATPase. To investigate structural changes within the Ca-ATPase that result from the phosphorylation of PLB by cAMP-dependent protein kinase (PKA), we have covalently bound the long-lived phosphorescent probe erythrosin isothiocyanate (Er-ITC) to cytoplasmic sequences within the Ca-ATPase. Under these labeling conditions, the Ca-ATPase remains catalytically active, indicating that observed changes in rotational dynamics reflect normal conformational transitions.
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J Biol Chem
March 1998
URA 2096 (CNRS) and Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France.
Treatment of rabbit sarcoplasmic reticulum Ca2+-ATPase with a variety of proteases, including elastase, proteinase K, and endoproteinases Asp-N and Glu-C, results in accumulation of soluble fragments starting close to the ATPase phosphorylation site Asp351 and ending in the Lys605-Arg615 region, well before the conserved sequences generally described as constituting the "hinge" region of this P-type ATPase (residues 670-760). These fragments, designated as p29/30, presumably originate from a relatively compact domain of the cytoplasmic head of the ATPase. They retain two structural characteristics of intact Ca2+-ATPase as follows: high sensitivity of peptidic bond Arg505-Ala506 to trypsin cleavage, and high reactivity of lysine residue Lys515 toward the fluorescent label fluorescein 5'-isothiocyanate.
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April 1992
Pharmacological Institute, Vienna, Austria.
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