Coupling of calcium transport with ATP hydrolysis in scallop sarcoplasmic reticulum.
Previously, we showed that incubation of the scallop sarcoplasmic reticulum (SR) with EGTA at above 37 degrees C resulted in the uncoupling of ATP hydrolysis with Ca2+ transport [Nagata et al. (1996) J. Biochem. 119, 1100-1105]. We have extended this study by comparing the kinetic behavior of Ca2+ release and binding to the uncoupled SR with that of intact scallop or rabbit SR. The change in the Ca2+ concentration in the reaction medium, as determined as the absorption of APIII, was followed using a stopped flow system. Intact scallop SR was preincubated with Ca2+ in the presence of a Ca2+ ionophore, A23187, and then ATP was added to initiate the reaction. The Ca2+ level in the medium increased to the maximum level in several seconds, and then slowly decreased to the initial low level. The rising and subsequent slow decay phases could be related to the dissociation and reassociation of Ca2+ with the Ca-ATPase, respectively. When uncoupled scallop SR vesicles were preincubated with CaCl2 in the absence of A23187 and then the reaction was initiated by the addition of ATP, a remarkable amount of Ca2+ was released from the SR vesicles into the cytosolic solution, whereas, with intact scallop or rabbit SR, only a sharp decrease in the Ca2+ level was observed. Based on these findings, we concluded that the heat treatment of scallop SR in EGTA may alter the conformation of the Ca-ATPase, thereby causing Ca2+ to be released from the enzyme, during the catalytic cycle, at the cytoplasmic surface, but not at the lumenal surface of SR vesicles.
Matsuo
Naoko
N
Department of Biology, Graduate School of Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
Nagata
Yoshiaki
Y
Nakamura
Jun
J
Yamamoto
Taibo
T
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006358:Hot Temperature; D006868:Hydrolysis / Q000187:drug effects; D008974:Mollusca; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D000001:Calcimycin; D014638:Vanadates; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium