Substrate regulation of calcium binding in Ca2+-ATPase molecules of the sarcoplasmic reticulum. II. Effect of CTP, GTP, ITP, and UTP.
To examine the effect of CTP, GTP, ITP, and UTP on calcium binding of Ca2+-ATPase molecules of the sarcoplasmic reticulum, the calcium dependence of the Ca2+-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 approximately 1, apparent calcium affinity (K(0.5)) of 2-6 microm)) and cooperative (n(H) approximately 2, K(0.5) approximately 0.2 microm) calcium binding of the molecules, whereas UTP caused the molecules to highly cooperatively (n(H) approximately 4) bind calcium ions with a lowered K(0.5) of approximately 0.04 microm. 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 microm), although the effect of the NTPs on the negatively cooperative manner (n(H) approximately 0.5) of calcium binding was not experimentally obtained. Taking into account the first part of this study (Nakamura, J., Tajima, G., Sato, C., Furukohri, T., and Konishi, K. (2002) J. Biol. Chem. 277, 24180-24190) showing the improving effect of ATP on calcium binding of the membranous and solubilized molecules, the results show that ATP is the only intrinsic substrate for the enzyme molecule. This NTP regulation is discussed in terms of the oligomeric structure of the molecules.
Nakamura
Jun
J
Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578, Japan. jun-n@mail.cc.tohoku.ac.jp
Tajima
Genichi
G
Sato
Chikara
C
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D007293:Inosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014544:Uridine Triphosphate / Q000494:pharmacology*
D007293:Inosine Triphosphate; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000252:Calcium-Transporting ATPases; D014544:Uridine Triphosphate