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Table 3.

Simulations of possible results of experiments to help assess if Ca was “lost” to parvalbumin

 
  Case 1: No Mg and no parvalbumin Case 2: No Mg and with parvalbumin Case 3: With both Mg and parvalbumin 
  Muscle solution With EGTA Muscle solution With EGTA Muscle solution With EGTA 
(all concentrations in µM
Rows 1–5 are assumed (typical or expected total values for sample) 
CaT 21.234 21.234 21.234 21.234 21.234 21.234 
BAPTAT 120.0 120.0 120.0 120.0 120.0 120.0 
EGTAT  1,000.0  1,000.0  1,000.0 
ParvT   7.0 7.0 7.0 7.0 
MgT     2,000.0 2,000.0 
Ca2+ and Mg2+ values adjusted to give CaT and MgT values above 
Ca2+ 0.0472 8.9 × 10−5 0.0339 8.9 × 10−5 0.0495 0.000149 
Mg2+     1,983.893 1590.948 
Rows 8–17 determined with above values for Ca2+ and Mg2+ 
BAPTA 98.8132 119.9513 103.9706 119.9514 89.449 109.663 
10 CaBAPTA 21.1868 0.0487 16.0294 0.0486 20.113 0.074 
11 MgBAPTA     10.439 10.263 
12 EGTA  978.8149  978.8664  586.676 
13 CaEGTA  21.1851  21.1336  21.152 
14 MgEGTA      392.173 
15 Parvalbumin   1.8293 6.9484 0.260 0.379 
16 CaParvalbumin   5.1707 0.0516 1.072 0.005 
17 MgParvalbumin     5.668 6.617 
Differences between values with and without EGTA 
18 ΔBAPTA  21.1381  15.9808  20.214 
19 ΔCaBAPTA  −21.1381  −15.9808  −20.039 
20 ΔMgBAPTA      −0.176 
Rows 21–24: Absorbance changes at 292 nm calculated from rows 18–20 
21 ΔABAPTA  0.1059  0.0800  0.1013 
22 ΔACaBAPTA  −0.0067  −0.0051  −0.0064 
23 ΔAMgBAPTA      −0.0005 
24 ΔAtotal or A0-AM  0.0992  0.0749  0.0944 
Predicted [CaT] = (A0AM)/(Δε · l) 
25 Predicted CaT  21.151  15.970  20.132 
Row 26 gives the value in row 25 divided by that in row 1 
26 Predicted/actual  0.996  0.752  0.948 
 
  Case 1: No Mg and no parvalbumin Case 2: No Mg and with parvalbumin Case 3: With both Mg and parvalbumin 
  Muscle solution With EGTA Muscle solution With EGTA Muscle solution With EGTA 
(all concentrations in µM
Rows 1–5 are assumed (typical or expected total values for sample) 
CaT 21.234 21.234 21.234 21.234 21.234 21.234 
BAPTAT 120.0 120.0 120.0 120.0 120.0 120.0 
EGTAT  1,000.0  1,000.0  1,000.0 
ParvT   7.0 7.0 7.0 7.0 
MgT     2,000.0 2,000.0 
Ca2+ and Mg2+ values adjusted to give CaT and MgT values above 
Ca2+ 0.0472 8.9 × 10−5 0.0339 8.9 × 10−5 0.0495 0.000149 
Mg2+     1,983.893 1590.948 
Rows 8–17 determined with above values for Ca2+ and Mg2+ 
BAPTA 98.8132 119.9513 103.9706 119.9514 89.449 109.663 
10 CaBAPTA 21.1868 0.0487 16.0294 0.0486 20.113 0.074 
11 MgBAPTA     10.439 10.263 
12 EGTA  978.8149  978.8664  586.676 
13 CaEGTA  21.1851  21.1336  21.152 
14 MgEGTA      392.173 
15 Parvalbumin   1.8293 6.9484 0.260 0.379 
16 CaParvalbumin   5.1707 0.0516 1.072 0.005 
17 MgParvalbumin     5.668 6.617 
Differences between values with and without EGTA 
18 ΔBAPTA  21.1381  15.9808  20.214 
19 ΔCaBAPTA  −21.1381  −15.9808  −20.039 
20 ΔMgBAPTA      −0.176 
Rows 21–24: Absorbance changes at 292 nm calculated from rows 18–20 
21 ΔABAPTA  0.1059  0.0800  0.1013 
22 ΔACaBAPTA  −0.0067  −0.0051  −0.0064 
23 ΔAMgBAPTA      −0.0005 
24 ΔAtotal or A0-AM  0.0992  0.0749  0.0944 
Predicted [CaT] = (A0AM)/(Δε · l) 
25 Predicted CaT  21.151  15.970  20.132 
Row 26 gives the value in row 25 divided by that in row 1 
26 Predicted/actual  0.996  0.752  0.948 

This table provides simulations of experimentally predicted values of [CaT] with and without Mg present and with and without metal binding to parvalbumin. The text provides most of the details for the assumed and predicted values. For the case of parvalbumin, Ca and Mg binding was calculated as described with Table 2. Relationships used to calculate Ca and Mg binding to BAPTA and EGTA are given in Section 1 of the supplemental text. For rows 21 and 22, values of 5,009 and 320 M−1 cm−1, respectively, were used for the extinction coefficients at 292 nm of the metal-free and Ca-bound forms of BAPTA (εB and εCaB, respectively; see Materials and methods). The value of 2,665 M−1 cm−1 assumed for the extinction coefficient for the Mg-bound form of BAPTA (εMgB) in row 23 was based on the observation of Tsien (1980) that the decrease in absorbance of BAPTA with Mg binding was about half of that produced by Ca binding (2,665 = 320 + 0.5 × (5,009 − 320)). As used throughout, the value of Δε used to calculate [CaT] (row 25) from A0AM (row 24) was εCaB − εB, i.e., −4,689 M−1 cm−1 (−4,689 = 320 − 5,009). Row 26 gives the ratios of the values in row 25 to those in row 1.

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