Comparison between ATP-Supported and GTP-Supported Phosphate Turnover of the Calcium-Transporting Sarcoplasmic Reticulum Membranes

Abstract

The interrelationship of the phosphate-transferring activity of the Ca-transporting rabbit sarcoplasmic reticulum membrane vesicles was studied: the phosphate exchange between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP) (NTP-NDP exchange), the Ca-dependent NTPase and the phosphorylation of NDP by Pi in the presence of NTP (NTP-Pi exchange). Different nucleotides were used as phosphate donors and acceptors. For the phosphate transfer from ITP or GDP the NTP-NDP exchange exhibits ping-pong kinetics with Mg-ITP and unliganded GDP as substrates. The apparent affinities of the enzyme for the NDP and NTP species are deduced according to this mechanism. The enzyme''s affinity for the nucleoside triphosphates and diphosphates depends on its functional state being considerably lower under conditions of NTP-NDP exchange than during NTP splitting or NTP synthesis. ATP and GTP are split with the same low rates when Ca-activated NTPase is inhibited by high internal Ca concentrations after Ca transport has reached steady state. The rates of the NTP-NDP exchange reactions differ by a factor of about 10 being .apprxeq. 3 .mu.mol.cntdot.mg-1.cntdot.min-1 for ATP-ADP and only .apprxeq. 0.3 .mu.mol.cntdot.mg-1.cntdot.min-1 (22.degree. C) for GTP-GDP. When the sarcoplasmic reticulum vesicles are made Ca-permeable, the Ca transport ATPase is turned on the rates of GTP and ATP splitting increase about 10-fold. While the rate of ATP-ADP exchanges is little reduced, the rate of GTP-GDP exchange drops by approximately 50%. The persisting exchange activity of Ca-permeable vesicles demonstrates that high internal Ca concentrations are not required for the transfer of the protein-bound phosphoryl group to NDP during NTP-NDP exchange.