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TABLE II

Rate Constants for the Reactions shown in Fig. 2 (16 °C)

1
2
3
4
Reaction
Forward
Reverse
Ratio
A.    
    Ca2+ + ATP ⇋ CaATP *0.1364 × 108M−1s−1 30,000 s−1 2,200 μM 
B.    
    Ca2+ + Parv ⇋ CaParv 0.417 × 108M−1s−1 0.5 s−1 0.012 μM 
    Mg2+ + Parv ⇋ MgParv 0.33 × 105M−1s−1 3 s−1 91.0 μM 
C.    
    Ca2+ + Trop ⇋ CaTrop 1.77 × 108M−1s−1 1,544 s−1 8.72 μM 
    Ca2+ + CaTrop ⇋ Ca2Trop 0.885 × 108M−1s−1 17.1 s−1 0.194 μM 
D.    
    Ca2+ + E ⇋ CaE 1.74 × 108 M−1 s−1 6.97 s−1 0.04 μM 
    Ca2+ + CaE ⇋ Ca21.74 × 108 M−1 s−1 8.71 s−1 0.05 μM 
    Mg2+ + E ⇋ MgE 8.71 × 104 M−1 s−1 4.36 s−1 50 μM 
    Mg2+ + MgE ⇋ Mg28.71 × 104 M−1 s−1 87.1 s−1 1,000 μM 
    H+ + E ⇋ HE instantaneous instantaneous (pK=8) 
    H+ + HE ⇋ H2instantaneous instantaneous (pK=8) 
    H+ + H2E ⇋ H3instantaneous instantaneous (pK=6) 
    H+ + H3E ⇋ H4instantaneous instantaneous (pK=5) 
    Ca2E ⇋ E + (2 Ca2+3.48 s−1 
E.    
    Ca2+ + Dye ⇋ CaDye 2.33 × 108M−1s−1 11,416 s−1 49 μM 
    Pr + Dye ⇋ PrDye 0.100 × 108M−1s−1 3,660 s−1 366 μM 
    Ca2+ + PrDye ⇋ CaPrDye 0.466 × 108M−1s−1 4,909 s−1 105 μM 
    Pr + CaDye ⇋ CaPrDye 0.100 × 108M−1s−1 7,869 s−1 787 μM 
1
2
3
4
Reaction
Forward
Reverse
Ratio
A.    
    Ca2+ + ATP ⇋ CaATP *0.1364 × 108M−1s−1 30,000 s−1 2,200 μM 
B.    
    Ca2+ + Parv ⇋ CaParv 0.417 × 108M−1s−1 0.5 s−1 0.012 μM 
    Mg2+ + Parv ⇋ MgParv 0.33 × 105M−1s−1 3 s−1 91.0 μM 
C.    
    Ca2+ + Trop ⇋ CaTrop 1.77 × 108M−1s−1 1,544 s−1 8.72 μM 
    Ca2+ + CaTrop ⇋ Ca2Trop 0.885 × 108M−1s−1 17.1 s−1 0.194 μM 
D.    
    Ca2+ + E ⇋ CaE 1.74 × 108 M−1 s−1 6.97 s−1 0.04 μM 
    Ca2+ + CaE ⇋ Ca21.74 × 108 M−1 s−1 8.71 s−1 0.05 μM 
    Mg2+ + E ⇋ MgE 8.71 × 104 M−1 s−1 4.36 s−1 50 μM 
    Mg2+ + MgE ⇋ Mg28.71 × 104 M−1 s−1 87.1 s−1 1,000 μM 
    H+ + E ⇋ HE instantaneous instantaneous (pK=8) 
    H+ + HE ⇋ H2instantaneous instantaneous (pK=8) 
    H+ + H2E ⇋ H3instantaneous instantaneous (pK=6) 
    H+ + H3E ⇋ H4instantaneous instantaneous (pK=5) 
    Ca2E ⇋ E + (2 Ca2+3.48 s−1 
E.    
    Ca2+ + Dye ⇋ CaDye 2.33 × 108M−1s−1 11,416 s−1 49 μM 
    Pr + Dye ⇋ PrDye 0.100 × 108M−1s−1 3,660 s−1 366 μM 
    Ca2+ + PrDye ⇋ CaPrDye 0.466 × 108M−1s−1 4,909 s−1 105 μM 
    Pr + CaDye ⇋ CaPrDye 0.100 × 108M−1s−1 7,869 s−1 787 μM 

Letters A–E correspond to the reactions in panels A–E of Fig. 2, where forward (column 2) and reverse (column 3) rate constants are denoted by k+i and k-i (i = 1, 2, etc.), respectively. Column 4 gives the dissociation constant (=k−i/k+i). The rate constants were obtained by scaling those of Hollingworth et al. (2006) at 18°C by the factor 0.871, which corresponds to a Q10 of 2. In A, the asterisk in column 2 denotes an apparent rate constant at free [Mg2+] = 1 mM, [K+] = 0.15 M, pH = 7, and viscosity = 2 centipoise (see Baylor and Hollingworth, 1998). In D, the dissociation constants of the reactions with H+ are expressed as pKs (shown in parentheses); the parentheses on the righthand side of the last reaction in column 1 denote the two Ca2+ ions transported by the pump to the lumen of the SR. In E, “Dye” denotes furaptra and “Pr” denotes protein sites capable of binding furaptra. Steady-state calculations with the scheme in E show that with the protein concentration in Table I, the relation between free [Ca2+] and the fraction of furaptra in the Ca2+-bound form (ΔfCaD = ([CaDye]+[CaPrDye])/[DyeTotal]) is very accurately described by a 1:1 binding equation with an apparent KD of 70 μM, the value estimated for frog myoplasm under steady-state conditions (Baylor and Hollingworth, 1998). However, when the peak of Δ[Ca2+] elicited by an AP is calculated from ΔfCaD in a single-compartment calibration (Eq. 3), a larger KD, 96 μM, is required (see Materials and Methods and Baylor and Hollingworth, 1998).

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