Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum at Glu-439 with 5-(bromomethyl)fluorescein.
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.
Stefanova
H I
HI
Department of Biochemistry, University of Southampton, U.K.
Mata
A M
AM
Gore
M G
MG
East
J M
JM
Lee
A G
AG
D000595:Amino Acid Sequence; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry*; D005452:Fluoresceins; D005971:Glutamates; D018698:Glutamic Acid; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D005971:Glutamates; D010446:Peptide Fragments; C081567:5-(bromomethyl)fluorescein; D018698:Glutamic Acid; D017301:Ca(2+) Mg(2+)-ATPase