Specific Association of Bromocresol Purple Anions with a Magnesium Complex of a Phosphorylated Intermediate During Steady-State Hydrolysis of ATP by the Mg²⁺+Ca²⁺-Dependent ATPase of Sarcoplasmic Reticulum

Abstract

The mechanism of dimeric binding of bromocresol purple (BCP) anions to Mg²⁺+Ca²⁺-ATPase of the sarcoplasmic reticulum (SR) and the resulting partial inhibition of the ATPase activity were studied.
BCP anions in three states, free monomer, bound monomer, and bound dimer, were spectrophotometrically calculated by solving simultaneous equations, ΔA_??_₁-_??_₂=∑Δα_i(ε₁^λ₁-ε₁^λ₁), and concentration changes of these states were analyzed. The addition of ATP caused an increase in the bound dimer and a decrease in the free monomer, but the change of the bound monomer was slight. The decrease in ΔA (decrease phase) on the addition of ATP on dual-wavelength spectrophotometry at 585-610 nm was related to an increase in the amount of dimer bound to the SR membranes. The magnitude of the decrease phase increased with an increase in Mg²⁺ concentration and decreased with an increase in the concentration of Ca²⁺.
BCP anions at the probe concentration partially inhibited the ATPase activity, and brought about a decrease in the ADP-sensitive E-P (E₁P) and an increase in the ADP-insensitive E-P (E₂P), though BCP anions did not affect the amount of total E-P. On elimination of Mg²⁺ at the steady-state E-P level both E₂P and E₂P•(BCP)₂ were decomposed, suggesting that the enzyme form binding the BCP dimer was Mg•E-P. An increase in Mg²⁺ concentration increased E₂P but an increase in Ca²⁺ concentration decreased E₂P. Decomposition of E₂P to P₁ was inhibited by BCP anions. The following simple scheme was suggested to explain the partial inhibition of the ATPase activity,
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Application of BCP anions was discussed for use as a probe for Mg•E-P in the steady-state ATP hydrolysis.