Oxidative phosphorylation and related reactions in particulate fractions from insects Public Deposited

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  • Oxidative phosphorylation and related reactions, particularly as affected by 2, 4-dinitrophenol (DNP), were studied with mitochondria and submitochondrial particles isolated from the flight muscle of the blowfly (Phormia regina) and housefly (Musca domestida). In the presence of a phosphate acceptor, the mitochondria oxidized pyruvate rapidly, and this was tightly coupled to phosphorylation. Added succinate and other citric acid cycle intermediates were not readily oxidized by the intact mitochondria. However, submitochondrial particles coupled succinate or NADH oxidation to phosphorylation, but did not utilize pyruvate. The substrate specificity of intact mitochondria appears to be related to a membrane permeability barrier. Pyruvate oxidation was stimulated by DNP, but only in the presence of ATP (or ADP) and Pi. DNP inhibited the ATP-Pi exchange reaction and promoted ATP hydrolysis with no substrate present. However, with sufficient ATP and Pi-Pi³² added, little or no net ATP hydrolysis occurred when pyruvate oxidation was stimulated by DNP, and ATP³² continued to be formed. The ATP (or ADP) and Pi requirements are due to their need in substrate-level phosphorylation because DNP still promoted respiration (in the presence of ATP, ADP, and Pi) after coupled phosphorylation and DNP-ATPase were completely inhibited by oligomycin. In the presence of oligomycin, DNP stimulated respiration, with ATP and Pi added, only when sufficient MgC1₂ (2 mM) was present to provide ADP for substrate-level phosphorylation. MgC1₂, however, did not promote respiration in the presence of oligomycin and in the absence of DNP, and MgC1₂ was not essential when ADP was present. These findings show that ATP (or ADP) and Pi are not obligatory in the basic mechanism by which DNP promotes electron transport in insect mitochondria; they also show that DNP can 'release' respiration at all three sites of coupled phosphorylation in the presence of oligomycin. However, at 0.1 to 0.15 mM DNP, maximal respiratory stimulation was obtained only in the absence of oligomycin, when DNP could promote ATP hydrolysis and uncouple phosphorylation. ATP³² formation from oxidative phosphorylation was demonstrated in experiments in which respiration was stimulated nearly maximally by 0.1 mM DNP in the presence of ATP and Pi-Pi³². Other experiments, which utilized ADP, or ATP and hexokinase, as a phosphate acceptor, indicated that the equivalent of two phosphorylation sites were not completely uncoupled by 0.1 mM DNP, since P/O ratios significantly greater than 1 were obtained with short incubation periods, even when the phosphate acceptor was not added until 10 minutes after the DNP. These results suggest that DNP does not 'release' respiration equally at each of the three sites of coupled phosphorylation. In contrast to mitochondria, sonic or digitonin particles did not show ATP-Pi exchange or DNP-ATPase activity. Sonic particles coupled succinate or NADH oxidation to phosphorylation with P/O ratios between 0.2 and 0.8; the phosphorylation was inhibited by oligomycin and uncoupled by DNP. Therefore, DNP can uncouple respiration in one or more reactions that do not necessarily lead to ATP hydrolysis. Mg⁺⁺-ATPase was observed with both mitochondria and particle preparations. At 0.4 mM, DNP caused complete inhibition of pyruvate oxidation and coupled phosphorylation with mitochondria, but did not inhibit succinate or NADH oxidation with sonic particles, although it did uncouple phosphorylation completely.
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