To examine the effect of CTP, GTP, ITP, and UTP on calcium binding of Ca 2؉ -ATPase molecules of the sarcoplasmic reticulum, the calcium dependence of the Ca 2؉ -activated hydrolysis activities of these NTPs of the enzyme molecules was examined by comparison with that of calcium binding of the molecules in the absence of the NTPs at pH 7.40. In the sarcoplasmic reticulum membrane, CTP, GTP, and ITP did not affect the noncooperative (Hill value (n H ) of ϳ1, apparent calcium affinity (K 0.5 ) of 2-6 M)) and cooperative (n H ϳ 2, K 0.5 ϳ 0.2 M) calcium binding of the molecules, whereas UTP caused the molecules to highly cooperatively (n H ϳ 4) bind calcium ions with a lowered K 0.5 of ϳ0.04 M. When the enzyme molecules were solubilized with detergent, all of these NTPs reversibly degraded the calcium affinity of the molecule (from K 0.5 ؍ 3-5 to >40 M), although the effect of the NTPs on the negatively cooperative manner (n H ϳ 0.5) of calcium binding was not experimentally obtained. Taking into account the first part of this study (Nakamura, J., Tajima The Ca 2ϩ -ATPase of the sarcoplasmic reticulum (SR) 1 belongs to the cation-transporting P-type ATPase family that is phosphorylated by ATP (1, 2), and Ca 2ϩ -ATPase catalyzes transport of 2 mol of calcium ion across the SR membrane, which is coupled with hydrolysis of 1 mol of ATP by the ATPase (3-5). This ATP hydrolysis reaction coincides with the transition of the enzyme state of the ATPase from E 1 (high affinity state for the calcium ion) to E 2 (low affinity state for the calcium ion). E 1 binds two calcium ions, and calcium-bound E 1 (E 1 P) is phosphorylated with ATP. Phosphorylated E 1 is ADPreactive and forms ATP with added ADP. E 1 P converts to its ADP-nonreactive form (E 2 P). After dephosphorylation of E 2 P, E 2 changes back to E 1 . This hydrolysis cycle has been shown to be accelerated by ATP binding to a putative regulatory site(s) (3, 5-7) at concentrations higher than those required for saturation of the catalytic site (8 -13). Ca 2ϩ -ATPase has been found to also utilize NTPs other than ATP (CTP, GTP, ITP, and UTP) (9, 14 -17). Of these NTPs, GTP and ITP have also been observed to have such a regulatory effect on NTP hydrolysis (9, 15, 16), whereas UTP has not been shown to have such an effect (17). However, CTP, GTP, ITP, and UTP have been shown to be able to support calcium transport (14).In the calcium transport reaction, calcium binding to the two calcium transport sites of the enzyme is a crucial step for driving the reaction, and such binding is required for phosphorylation of the enzyme with ATP (3-5), as mentioned above. In the first part of this study on substrate regulation of calcium binding (32), the regulatory effect of ATP on calcium binding was examined, and ATP binding to the regulatory site of the enzyme was found to raise the calcium binding performance of the enzyme molecules depending on the ATP concentration, pH, and aggregation state of the molecules. Here, we examined the regulatory effects of CTP, GTP, ITP, and UTP on...