A fluorescence study of the kinetics of the sarcoplasmic reticulum Ca2+-ATPase

Henderson, Ian Matthew John (1993) A fluorescence study of the kinetics of the sarcoplasmic reticulum Ca2+-ATPase. University of Southampton, Doctoral Thesis.

Abstract

The effects of pH, Mg²⁺ and Ca⁺² upon boththe intrinsic tryptophan fluorescence of the Ca²⁺-ATPase, and the fluorescence of the Ca²⁺-ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl), were used to study the binding of Ca²⁺ to the Ca²⁺-ATPase. The tryptophan fluorescence of the Ca²⁺-ATPase was sensitive to the calcium occupancy of the high affinity Ca²⁺ binding sites of the Ca²⁺-ATPase and it is proposed that this is due to a conformational change occurring between binding of the first and second Ca²⁺ ions. The fluorescence of NBD-Ca²⁺-ATPase was shown to be sensitive to pH, Mg²⁺ and Ca²⁺ and is proposed to be sensitive to the E2-E1 transition.

Computer modelling of the effects of pH, Mg²⁺ and Ca²⁺ upon both the intrinsic tryptophan fluorescence of the Ca²⁺-ATPase, and the fluorescence of NBD-Ca²⁺-ATPase at equilibrium, together with simulations of stop-flow experimental data has allowed the development of a comprehensive kinetic model for Ca²⁺ binding to the Ca²⁺-ATPase, and the effects of pH, Mg²⁺ and Ca²⁺ concentration on thisbinding. The use of the fluorescent nucleotide analogue 2'3'-0-(2,4,6-trinitrophenyl)-adenosine dephosphate (TNP-ADP), and the effect of ATP on the fluorescence of NBD-Ca²⁺-ATPase has allowed the model developed above to be expanded to include ATP binding.

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