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Labeling the (Ca(2+)-Mg2+)-ATPase of Sarcoplasmic Reticulum With 4-(bromomethyl)-6,7-dimethoxycoumarin: Detection of Conformational Changes

Labeling the (Ca(2+)-Mg2+)-ATPase of Sarcoplasmic Reticulum With 4-(bromomethyl)-6,7-dimethoxycoumarin: Detection of Conformational Changes

H I Stefanova, J M East, M G Gore, A G Lee

Biochemistry. 1992 Jul 7;31(26):6023-31.

PMID: 1385723

Abstract:

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+.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP88404255	4-Bromomethyl-6,7-dimethoxycoumarin		88404-25-5	Price

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