Labeling the calcium-magnesium-ATPase of sarcoplasmic reticulum at Glu-439 with 5-(bromomethyl)fluorescein

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.