JBC, Vol. 250, Issue 18, 7219-7224, Sep, 1975
Transport and inhibitory Ca2+ binding sites on the ATPase enzyme isolated from the sarcoplasmic reticulum
N. Ikemoto
Ca2+ binding sites located on the Ca2+-dependent ATPase purified from the
fragmented sarcoplasmic reticulum (Ikemoto, N (1974) J. Biol. Chem. 249,
649) have been further studied. At 0 degrees there are three classes of
binding sites denoted as alpha (K congruent to 3 times 10(61 M-1), beta(K
congruent to 5 times 10(4) M-1), and gamma (K congruent to 1 times 10(3)
M-1) sites. At 22 degrees there is no beta site but there are about two
alpha sites per 10(5) daltons, while at 0 degrees there is one alpha and
one beta site. The change is reversible. The parallelism between the
temperature-induced changes in the alpha site and the reported (Sumida, M.,
and Tonomura, Y. (1974) J. Biochem. 75, 283) temperature dependence of the
ratio of Ca2+ transport and ATP cleavage (deltaCa2+/deltaATP is 2 at 22
degrees and 1 at 0 degrees) suggests the involvement of the alpha site in
transport. Studies at a low ATP to enzyme ratio (0.5 to 2.5 mol of
ATP/10(5) g of ATPase unit) permitting the separate investigation of the
phosphorylation and dephosphorylation process show that concomitantly with
the formation of the phosphorylated enzyme (E approximately P) bound
calcium is released from, and concomitantly with the dephosphorylation it
is rebound to, the alpha site. Binding of Ca2+ to the E approximately P
moiety inhibits the liberation of Pi. Analysis by use of a Hill plot of the
Ca2+ dependence of the inhibition suggests the involvement of two sites
with an average affinity of approximately 10(3) M-1. These have tentatively
been identified as alpha (low affinity form) and gamma sites.
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