Unexpected phosphoryl transfer from Asp351 to fluorescein attached to Lys515 in sarcoplasmic reticulum Ca2+-ATPase.
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.
McIntosh
David B
DB
Institute of Infectious Diseases and Molecular Medicine, Division of Chemical Pathology, and National Health Laboratory Services, University of Cape Town, South Africa. David.McIntosh@uct.ac.za
Montigny
Cédric
C
Champeil
Philippe
P
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D002384:Catalysis; D004795:Enzyme Stability; D019793:Fluorescein / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000378:metabolism*; D010755:Organophosphates / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D010755:Organophosphates; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D001224:Aspartic Acid; C011632:acetyl phosphate; D000469:Alkaline Phosphatase; D014357:Trypsin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D008239:Lysine; D019793:Fluorescein