Abstract
To measure the free intrasarcoplasmic reticulum Ca (CaSR) in isolated
rat cardiac microsomes, ventricular tissue was homogenized in the
presence of the low-affinity Ca indicator furaptra. Stepwise increases
in cuvette Ca (Cac) in the presence of ATP caused progressive
increases in steady-state intravesicular fluorescence ratio to a
maximum (Rmax). Steady-state CaSR/Cac was approximately 7000.
Therefore the resting CaSR may approach 700 microM in the rat cardiac
myocyte at Cac = 100 nM. The sarcoplasmic reticulum (SR) Ca pump
requires a free energy of deltaG approximately 44 kJ x mol(-1) to
generate this Ca gradient (e.g., approximately 74\% of deltaG(ATP)).
Total SR 45Ca uptake was also measured in digitonin-permeabilized
myocytes as a function of Cac in the absence of precipitating ions.
The steady-state SR Ca content at 100 nM Cac was approximately
400 micromol/liter cytosolic volume. Used together, these data allowed
evaluation of the in situ SR Ca-buffering properties. The SR Ca-binding
site concentration was approximately 14 mM, and Kd(Ca) approximately
0.638 mM CaSR.
- 9284319
- adenosine
- animals,
- atpase,
- calcium,
- calorimetry,
- fluorescence,
- heart
- kinetics,
- microsomes,
- myocardium,
- rats,
- reticulum,
- sarcoplasmic
- spectrometry,
- sprague-dawley,
- thermodynamics,
- triphosphate,
- ventricles,
- {c}a$^{2+}$-transporting
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