J. Biol. Chem., Vol. 261, Issue 4, 1495-1498, Feb, 1986
Occluded calcium sites in soluble sarcoplasmic reticulum Ca2+-ATPase
MR Klemens, JP Andersen and CM Grisham
Rabbit muscle sarcoplasmic reticulum Ca2+-ATPase has been shown to bind
gadolinium ion (Gd3+) at two high affinity Ca2+ sites (Stephens, E. M., and
Grisham, C. M. (1979) Biochemistry 18, 4876-4885). Gd3+ bound at these
sites exhibits an unusually long electron spin relaxation time, consistent
with occlusion of these sites and reduced contact with solvent H2O. In this
report, the nature of the Gd3+ sites was examined in preparations of the
enzyme solubilized with the detergent C12E8. The frequency dependence of
water proton relaxation in solutions containing the solubilized Ca2+-ATPase
yields dipolar correlation times, tau c, for the 1H-Gd3+ interaction of
1.04 X 10(-9) s for Gd3+ bound at site 1 and 1.98 X 10(-9) s for Gd3+ bound
at site 2. The correlation time itself is frequency dependent below 30 MHz,
indicating that the correlation time is dominated by the electron spin
relaxation time of bound Gd3+. The long values of the correlation time
found in the present study are consistent with a poor accessibility of
these Gd3+ sites (particularly site 2) to solvent water molecules.
Analytical ultracentrifugation and molecular sieve high performance liquid
chromatography indicated that the active fraction of the soluble Ca2+-
ATPase was monomeric. Thus occlusion of the Ca2+ sites in this enzyme is
largely dependent on the tertiary structure of the monomeric ATPase and
does not appear to depend on multimeric membrane structures.
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