Labeling the calcium-magnesium-ATPase of sarcoplasmic reticulum at Glu-439 with 5-(bromomethyl)fluorescein
- 15 June 1993
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 32 (23) , 6095-6103
- https://doi.org/10.1021/bi00074a022
Abstract
The (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum was labeled with 5-(bromomethyl)fluorescein. A stoichiometry of one label per ATPase molecule was found, which was unaffected by the presence of ATP. Labeling resulted in a 60% decrease in ATPase activity. Sequencing identified the labeled residue as Glu-439. The fluorescence emission spectrum of the labeled ATPase was unaffected by the addition of Ca2+ or vanadate or by phosphorylation with either Pi or ATP. Measurement of the pK of the bound fluorescein and observation of quenching by KI were consistent with a relatively exposed location for the fluorophore. Measurements of fluorescence energy transfer located the position of Glu-439 relative to Lys-515 and Cys-344 and relative to the membrane surface. None of these distances changed in binding Ca2+ or vanadate.Keywords
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