Probing the nucleotide-binding site of sarcoplasmic reticulum (Ca2+-Mg2+)-ATPase with anti-fluorescein antibodies.
Antibodies raised against fluorescein were unable to bind to the fluorophore when bound at the nucleotide-binding site of native (Ca2+-Mg2+)-ATPase, as judged by fluorescence quenching assays or competitive ELISAs, but were able to bind when the ATPase was denatured. Indirect ELISAs, in which native and denatured FITC-ATPase were used to coat ELISA plates, were unable to detect the difference in accessibility of the fluorescein bound to the native and denatured ATPase. These results indicate that the nucleotide-binding site is relatively inaccessible in the native structure, even though fluorescence energy transfer studies [(1987) Biochim. Biophys. Acta 897, 207-216] indicate that this site must be close to the surface of the ATPase. In addition the results suggest that the indirect ELISA method may be of limited value in probing the accessibility of epitopes using antibodies.
Mata
A M
AM
Department of Biochemistry, University of Southampton, England.
Lee
A G
AG
East
J M
JM
D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D004797:Enzyme-Linked Immunosorbent Assay; D019793:Fluorescein; D005452:Fluoresceins / Q000276:immunology; D066298:In Vitro Techniques; D011487:Protein Conformation; D011489:Protein Denaturation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D019793:Fluorescein