Labelling the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum with o-phthalaldehyde (OPA) results in loss of ATPase activity at a 1:1 molar ratio of label to ATPase. The affinity of the ATPase for Ca2+ is unaffected, as is the E1/E2 equilibrium constant. The rate of dissociation of Ca2+ from the Ca2+-bound ATPase is also unaffected and Mg2+ increases the rate of dissociation, as for the unlabelled ATPase. Effects of Mg2+ on the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are also unaffected by labelling with OPA, consistent with the fluorescence change reporting on Mg2+ binding at the gating site on the ATPase. The affinity of the ATPase for ATP is reduced by labelling, as is the rate of phosphorylation. The rate of phosphorylation is independent of the concentration of ATP above 25 μM ATP, so that the slow step is the first-order rate constant for phosphorylation by bound ATP. The rate of the back reaction between phosphorylated ATPase and ADP is little affected, suggesting that the slow step in phosphorylation could be the slow conformation step before phosphoryl transfer. The rate of dephosphorylation of the phosphorylated ATPase is also decreased, suggesting that a similar conformation change could be involved in the dephosphorylation step. The rate of the Ca2+ transport step appears to be unaffected by labelling. The net result of these changes is that the labelled ATPase is present predominantly in a Ca2+-free, phosphorylated form at steady state in the presence of ATP.
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July 1996
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Research Article|
July 15 1996
The mechanism of inhibition of the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum by the cross-linker o-phthalaldehyde
Yamin M. KHAN
;
Yamin M. KHAN
1Department of Biochemistry and Institute for Biomolecular Sciences, University of Southampton, Southampton SO16 7PX, U.K.
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Anthony P. STARLING
;
Anthony P. STARLING
1Department of Biochemistry and Institute for Biomolecular Sciences, University of Southampton, Southampton SO16 7PX, U.K.
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J. Malcolm EAST
;
J. Malcolm EAST
1Department of Biochemistry and Institute for Biomolecular Sciences, University of Southampton, Southampton SO16 7PX, U.K.
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Anthony G. LEE
Anthony G. LEE
1Department of Biochemistry and Institute for Biomolecular Sciences, University of Southampton, Southampton SO16 7PX, U.K.
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Biochem J (1996) 317 (2): 439–445.
Article history
Received:
January 18 1996
Revision Received:
March 08 1996
Accepted:
March 18 1996
Citation
Yamin M. KHAN, Anthony P. STARLING, J. Malcolm EAST, Anthony G. LEE; The mechanism of inhibition of the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum by the cross-linker o-phthalaldehyde. Biochem J 15 July 1996; 317 (2): 439–445. doi: https://doi.org/10.1042/bj3170439
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