Guanosine triphosphate utilization by canine cardiac muscle sarcoplasmic reticulum.
We investigated the reaction mechanism for GTP-dependent Ca2+ uptake by canine cardiac microsomes enriched in fragmented sarcoplasmic reticulum (SR), because previous studies reported that GTP utilization in cardiac SR occurs via a pathway very different from that for ATP utilization (for a review, see "Entman, M.L., Bick, R., Chu, A., Van Winkle, W.B., & Tate, C.A. (1986) J. Mol. Cell. Cardiol. 18, 781-792"). In cardiac microsomes, we detected slow but distinct oxalate-dependent Ca2+ accumulation, which reached 550 nmol/mg protein in 10 min, and similarly slow Ca2+-dependent GTP hydrolysis. In 50 microM [gamma-32P]-GTP at 0 degrees C, we detected Ca2+-dependent formation of phosphoprotein whose level in the steady state was about a half of the maximum obtained with [gamma-32P]ATP. Kinetic properties of the phosphoprotein, its molecular weight and its chemical stability after the acid treatment are consistent with the conclusion that the phosphoprotein is an acylphosphate intermediate for Ca2+-dependent GTP hydrolysis catalyzed by the Ca2+-pump ATPase. Analysis of the kinetics of the turnover of phosphoprotein revealed that slow GTP hydrolysis is due to slow phosphoprotein formation; at 25 degrees C, the latter arises mainly from slow binding of Ca2+ to the dephosphorylated enzyme. These results indicate that, contrary to the previous data, the reaction pathway for GTP-dependent Ca2+ transport in cardiac SR is basically the same as that for ATP-dependent transport.
Ogurusu
T
T
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka.
Wakabayashi
S
S
Watanabe
T
T
Shigekawa
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D004285:Dogs; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D066298:In Vitro Techniques; D008861:Microsomes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D010750:Phosphoproteins; D006160:Guanosine Triphosphate; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases; D002118:Calcium