Reactivity of lysyl residues on the (Ca(2+)-Mg2+)-ATPase to 7-amino-4-methylcoumarin-3-acetic acid succinimidyl ester.
The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with the succinimidyl ester of 7-amino-4-methylcoumarin-3-acetic (AMCA). Although a large number of residues were labeled, it was found that Lys-492 was labeled preferentially at pH values between 6 and 8, consistent with an unusual environment for this residue. Labeling was reduced in the presence of ATP, suggesting that Lys-492 is in or near the ATP binding site of the ATPase. Other identified residues labeled by AMCA were Lys-35, Lys-135, Lys-218, Lys-371, and Lys-605. It is suggested that these represent surface-exposed lysyl residues. Lys-515, labeled by fluorescein isothiocyanate (FITC), was not labeled by AMCA. Labeling with AMCA at pH 6.0 has no effect on ATPase activity, suggesting that Lys-492 is not essential for activity. The fluorescence of AMCA-labeled ATPase did not change on addition of either ATP in the presence of Ca2+ or Pi in the absence of Ca2+, suggesting that Lys-492 was not affected by any major conformational changes on the ATPase. The efficiency of fluorescence energy transfer between AMCA and FITC labels on the ATPase was unaffected by binding Ca2+ or vanadate, arguing against any large-scale movement of the cytoplasmic domains of the ATPase.
Stefanova
H I
HI
Department of Biochemistry, University of Southampton, U.K.
Mata
A M
AM
East
J M
JM
Gore
M G
MG
Lee
A G
AG
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002118:Calcium / Q000494:pharmacology; D003374:Coumarins / Q000494:pharmacology; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry*; D008274:Magnesium / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D014638:Vanadates / Q000494:pharmacology
D003374:Coumarins; D005456:Fluorescent Dyes; D014638:Vanadates; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; D008239:Lysine; C051029:7-amino-4-methylcoumarin-3-acetic acid; D002118:Calcium