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Catecholamine Storage Organelles; Membrane Ghosts; Mg-AD P Evoked ΔμH·; ATP Synthesis; ATP-Pi Exchange
Abstract:
The reaction rates of ATP formation from ADP and inorganic phosphate (Pi) by the catecholamine storage organelles isolated from bovine adrenal medulla accelerated 5--6 fold, when Pi was added at various times after addition of Mg-ADP, as compared to the rates observed when the reaction partners were added simultaneously. The increase of the rates of ATP-Pi exchange upon subsequent addition of Pi to Mg-ATP was less prominent. Mg-ADP induced a delta psi (60--70 mV, positive inside), almost equal that induced by Mg-ATP. In both cases delta psi was significantly higher in the absence of Pi than in its presence. At pH 7.4 of the medium the delta pH was 1.4 units indicating an internal pH of 6. It was stable during the reaction time in the presence of Mg-ATP, while in the presence of Mg-ADP the internal pH increased gradually by 0.2 units. Both the ATP forming reactions as well as the nucleotide induced delta psi were uncoupler-sensitive (CCCP). Though inhibiting ATP formation from ADP + Pi by 50%, 0.1 mM AP5A (an inhibitor of adenylate kinase) did neither affect delta psi nor delta pH, hence ruling out the possibility that delta microH. induced by Mg-ADP would be actually due to AtP formed from ADP. Membrane ghosts were not able of ATP synthesis (in the absence of valinomycin), the ATP-Pi exchange was only 10--20% of that of intact organelles, due to the low delta microH. in the absence of intravesicular soluble constituents. It is proposed that the energy for ATP synthesis is furnished by the high gradients of protonated soluble constituents in the intact organelles.
