Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum with 4-(bromomethyl)-6,7-dimethoxycoumarin: detection of conformational changes.
The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It was shown that a single cysteine residue (Cys-344) was labeled on the ATPase, with a 25% reduction in steady-state ATPase activity and no reduction in the steady-state rate of hydrolysis of p-nitrophenyl phosphate. The fluorescence intensity of the labeled ATPase was sensitive to pH, consistent with an effect of protonation of a residue of pK 6.8. Fluorescence changes were observed on binding Mg2+, consistent with binding to a single site of Kd 4 mM. Comparable changes in fluorescence intensity were observed on binding ADP in the presence of Ca2+. Binding of AMP-PCP produced larger fluorescence changes, comparable to those observed on phosphorylation with ATP or acetyl phosphate. Phosphorylation with P(i) also resulted in fluorescence changes; the effect of pH on the fluorescence changes was greater than that on the level of phosphorylation measured directly using [32P]P(i). It is suggested that different conformational states of the phosphorylated ATPase are obtained at steady state in the presence of Ca2+ and ATP and at equilibrium in the presence of P(i) and absence of Ca2+.
Stefanova
H I
HI
Department of Biochemistry, SERC Centre for Molecular Recognition, University of Southampton, U.K.
East
J M
JM
Gore
M G
MG
Lee
A G
AG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism* / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D003374:Coumarins; D005456:Fluorescent Dyes; C053769:4-bromomethyl-6,7-dimethoxycoumarin; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D003545:Cysteine