Indications for an oligomeric structure and for conformational changes in sarcoplasmic reticulum Ca2+-ATPase labelled selectively with fluorescein.
Fluorescein isothiocyanate is a highly specific inhibitor of the Ca2+-ATPase from sarcoplasmic reticulum. The Ca2+ pumping is inhibited completely at a fluorescein isothiocyanate concentration half that of the ATPase protein, indicating that the protein is at least a dimer. ATP protected specifically against fluorescein isothiocyanate inhibition, indicating that fluorescein isothiocyanate may react at the nucleotide binding site of the ATPase (probably with a reactive lysine residue). The fluorescein is incorporated almost exclusively into the 105 kdalton catalytic polypeptide of the ATPase and digestion by trypsin gives rise to a fluorescein-labelled 45 kdalton fragment. Conformational changes induced by addition of Ca can be studied conveniently with the fluorescein-labelled ATPase.
D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013053:Spectrophotometry; D013861:Thiocyanates / Q000494:pharmacology*; D014357:Trypsin
D005452:Fluoresceins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D013861:Thiocyanates; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium