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

Abstract

The study deals with the interrelationship of the phosphate-transferring activities of the calciumtransporting sarcoplasmic reticulum membrane vesicles : the phosphate exchange between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP) (NTP-NDP exchange), the calcium-dependent NTPase, and the phosphorylation of NDP by inorganic phosphate in the presence of NTP (NTP-Pi exchange). Different nucleotides were used as phosphate donors and acceptors. It is demonstrated for the phosphate transfer from ITP to GDP that the NTP-NDP exchange exhibits ping-pong kinetics with Mg-ITP and unliganded GDP as substrates. The apparent affinities of the enzyme for the nucleoside diphosphate and triphosphate 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 calcium-activated NTPase is inhibited by high internal calcium concentrations after calcium transport has reached steady state. The rates of the NTP-NDP exchange reactions, however, differ by a factor of about 10 being E 3 pmol . mg-' . min-' for ATP-ADP and only E 0.3 pmol . mg-' . min-' (22°C) for GTP-GDP. When the sarcoplasmic reticulum vesicles are made calcium-permeable, the calcium transport ATPase is turned on and the rates of GTP and ATP splitting increase about tenfold. Yet, while the rate of ATP-ADP exchange is little reduced, the rate of GTP-GDP exchange drops by approximately 50 %. The persisting exchange activity of calcium-permeable vesicles demonstrates that high internal calcium concentrations are not required for the transfer of the protein-bound phosphoryl group to NDP during NTP-NDP exchange.

Unidirectional calcium and nucleotide fluxes in sarcoplasmic reticulum. I. Interpretation of flux ratios for different reaction schemes

“…It was found that ATP-ADP exchange is 4 to 10 times faster than the rate of calcium exchange (Waas and Hasselbach, 1981;Takenaka et al, 1982), while GTP-GDP exchange approximately matched calcium exchange (Ronzani et al, 1979). Ronzani et al (1979) therefore called into question the coupling of nucleotide diphosphokinase activity and calcium translocation. Takenaka and co-workers (1982) conclude ".…”

Excimer Formation of ATPase from Sarcoplasmic Reticulum Labeled with N‐(3‐Pyrene)maleinimide

“…Sarcoplasmic reticulum vesicles were prepared according to the procedure described by Hasselbach [31]. Calciuiii uptake was determined in the presence of 5 mM potassium oxalate by the Millipore filtration technique using the same buffer as described above, except A-231 87 was omitted.…”

Characterisation of the Enzyme Intermediates of the Sarcoplasmic Transport ATPase by the Use of Inhibitors

“…The rate of ATP-ADP-exchange is also inhibited at relatively high C a2+ con centrations [27] and the inhibition range depends on the Mg2+ concentration in the assay (Fig. 7).…”