LABELING THE (CA2-MG2+)-ATPASE OF SARCOPLASMIC-RETICULUM AT GLU-439 WITH 5-(BROMOMETHYL)FLUORESCEIN()

The (Ca2+-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum was l abeled with 5-(bromomethyl)fluorescein. A stoichiometry of one label p er ATPase molecule was found, which was unaffected by the presence of ATP. Labeling resulted in a 60% decrease in ATPase activity. Sequencin g 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 P(i) or ATP. Measuremen t of the pK of the bound fluorescein and observation of quenching by K I were consistent with a relatively exposed location for the fluoropho re. Measurements of fluorescence energy transfer located the position of Glu-439 relative to Lys-515 and Cys-344 and relative to the membran e surface. None of these distances changed on binding Ca2+ or vanadate .