J. Biol. Chem., Vol. 262, Issue 30, 14492-14497, 10, 1987
Calcium-dependent calcium occlusion in the sarcoplasmic reticulum Ca2+- ATPase. Its enhancement by phosphorylation of the enzyme
J Nakamura
Biological Institute, Faculty of Science, Tohoku University, Miyagi, Japan.
45Ca2+-40Ca2+ exchangeability of 45Ca bound to the calcium transport sites
of unphosphorylated sarcoplasmic reticulum Ca2+-ATPase at equilibrium has
been found to be heterogeneous: Half of the bound calcium is
[Ca2+]-dependent in a slowly exchangeable (k less than 0.3 s- 1),
"occluded" state in the Ca2+-ATPase, and the other calcium is
[Ca2+]-independent in a rapidly exchangeable (k approximately 0.3 s-1),
"unoccluded" state (Nakamura, J. (1986) Biochim. Biophys. Acta 870, 495-
501). In this paper, the two different forms of exchangeable calcium were
studied after phosphorylation of the enzyme by ATP without added Mg2+ at pH
7.0 and 0 degree C. By the phosphorylation, the degree of the occlusion
became higher (k less than 0.03 s-1). The unoccluded calcium was, however,
not significantly affected. The more highly occluded calcium exchanged at
the same rate as the decay rate of the phosphoenzyme (EP) in the steady
state at a ratio of about 1:1. The occluded calcium was relieved by
dephosphorylation of EP by ADP. These results suggest that 1 mol of
ADP-sensitive EP more highly occluded 1 mol of calcium, already occluded
before phosphorylation. After transformation of ADP-sensitive EP to its
ADP-insensitive form by the addition of 20 mM Mg2+ at pH 8.8, the
unoccluded calcium was rapidly (k = 0.1-0.3 s-1) released from the
transformed EP. However, the occluded calcium was maintained in an occluded
state in which the calcium was slowly (k approximately 0.01 s-1) released
from the EP without exchange. The results suggest that calcium occlusion in
the ADP- sensitive EP is not relieved by the loss of ADP sensitivity of the
EP itself.
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