| Summary: | <p>This thesis presents the results of studies on some aspects of oxidative phosphorylation, and of mitochondrial reactions which utilise the energy of oxidative phosphorylation.</p> <p><strong>The Stoichiometry of Oxidative Phosphorylation</strong></p> <ol type="1"> <li>ADP:O and P:O ratios of rat liver mitochondria (RLM) and heavy beef heart mitochondria (HBHM) were re-examined under a number of conditions, and by independent methods of measuring oxygen consumption and P<sub>1</sub> esterification.</li> <li>When appropriate precautions were taken ADP:O and P:O ratios obtained by all methods and under each of the conditions closely agreed, and did not exceed 3 for pyruvate plus L-malate, 2 for succinate, and 4 for α oxoglutarate oxidation.</li> <li>The high P:O ratios reported by Smith & Hansen (1964), Gurban & Cristea (1964, 1965) and Lynn & Brown (1965) were shown to be caused by an underestimation of oxygen consumption.</li> </ol> <p><strong>The Primary Phosphate Acceptor in Oxidative Phosphorylation</strong></p> <ol type="1"> <li>The nature of the nucleotide acceptor, to which P<sub>1</sub> is first linked in oxidative phosphorylation, was investigated by measuring the labelling by <sup>32</sup>P<sub>1</sub> of the bound adenine nucleotides of HBHM and phosphorylating submitochondrial particles (ETP<sub>H</sub>) during the first 2 min. of succinate oxidation.</li> <li>In all labelling experiments in the absence of uncouplers AT<sup>32</sup>P had a higher specific radioactivity than AD<sup>32</sup>P, and it was concluded that <em>ADP</em> is the primary <em>endogenous</em> acceptor of <sup>32</sup>P<sub>1</sub>. This disagrees with the results of Ozawa & MacLennan (1965), who claimed that AD<sup>32</sup>P contained most of the radioactivity in such experiments. Possible reasons for this discrepancy are discussed.</li> <li>In the presence of uncouplers, AD<sup>32</sup>P had a higher specific activity than AT<sup>32</sup>P in labelling experiments with HBHM. The labelling of AD<sup>32</sup>P was inhibited by arsenite, showing that it was caused by substrate-level phosphorylation. It is postulated that in substrate-level phosphorylation the sequence of labelling of endogenous nucleotides is GT<sup>32</sup>P → AD<sup>32</sup>P → AT<sup>32</sup>P, catalysed by succinate thiokinase, nucleoside monophosphate kinase and myokinase, as shown by Heldt & Schwalbach (1967) for rat liver mitochondria.</li> <li>The ability of AMP and ADP to stimulate the respiration of HBHM was investigated, and the phosphorylation of adenine nucleotides in such experiments was measured. It was found that AMP could only stimulate respiration in the presence of added Mg<sup>2+</sup>. In the absence of Mg<sup>2+</sup>, or if a concentration of EDTA greater than that of Mg<sup>2+</sup> was added, AMP was not phosphorylated. Under all the above conditions ADP stimulated the respiration of HBHM, and more than 90% of the ADP was converted to ATP at the State 3 → State 4 transition.</li> <li>It was concluded that <em>ADP</em> is the primary <em>external</em> phosphate acceptor in HBHM, and that AMP is only indirectly phosphorylated in oxidative phosphorylation via the myokinase reaction, which requires added Mg<sup>2+</sup>. The results of Ozawa (1966), who claimed that AMP was the primary phosphate acceptor, may be explained by wrong assumptions concerning the calculation of AMP:O ratios, and possible errors in the isolation and assay of adenine nucleotides.</li> </ol> <p><strong>The Purification of Coupling Factors, and the Effect of NAD<sup>+</sup> and NADH on the AD<sup>32</sup>P-ATP and <sup>32</sup>P<sub>i</sub>-ATP Exchange Reactions Catalysed by such Fractions</strong></p> <ol type="1"> <li>Attempts to purify a soluble factor from mitochondria, which was specific for oxidative phosphorylation at Site 1 in depleted sub-mitochondrial particles, were unsuccessful, but the 0–45% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> fraction of the supernatant from sonicated HBHM increased the P:O ratios of 'salt-extracted particles' (Green et al., 1963) and METP<sub>H</sub> (Linnane & Titchener, 1960) with NADH or succinate as substrate.</li> <li>This crude preparation of coupling factor had a high AD<sup>32</sup>P-ATP exchange activity, and a small <sup>32</sup>P<sub>i</sub>-ATP exchange activity which depended in a complex way on the relative concentrations of P<sub>i</sub>, ADP and ATP, and was inhibited by uncouplers or inhibitors of oxidative phosphorylation.</li> <li>The effect of NAD<sup>+</sup> and NADH on the AD<sup>32</sup>P-ATP and <sup>32</sup>P<sub>i</sub>-ATP exchange reactions catalysed by the 0–45% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> fraction was investigated over a wide range of experimental conditions, but no significant increases or decreases in activity were observed. These results are discussed in relation to the chemical theory of oxidative phosphorylation.</li> <li>The P<sub>i</sub>-ATP exchange activity of the crude coupling factor was further characterised, and was shown to be associated with a high molecular weight component containing cytochromes a, b and c, which could be spun down at 150,000 g for 1 hr. It was concluded that the P<sub>i</sub>-ATP exchange activity is associated with small fragments of the mitochondrial membrane and is not a soluble or homogenous enzyme.</li> <li>It was shown that although much of the AD<sup>32</sup>P-ATP exchange activity of the crude coupling factor was associated with myokinase, there was an excess of AD<sup>32</sup>P-ATP exchange activity which was possibly associated with the coupling factor.</li> </ol> <p><strong>The Mechanism of Penetration of Oxaloacetate and L-malate into Mitochondria</strong></p> <ol type="1"> <li>The penetration of oxaloacetate and L-malate into mitochondria was investigated by measuring the effect of oxaloacetate or L-malate on the concentration of intramitochondrial NADH, using the Aminco-Chance dual-wavelength spectrophotometer at 340–374 mμ.</li> <li>NADH oxidase was inhibited by rotenone plus antimycin A, and as the activity of the mitochondrial malate dehydrogenase under the test conditions was very high in relation to the rates of penetration of oxaloacetate and L-malate, the initial rate of oxidation of NADH was a measure of the rate of penetration of oxaloacetate, and the initial rate of reduction of NAD<sup>+</sup> was a measure of the penetration of L-malate.</li> <li>The rates of penetration of oxaloacetate and L-malate were greatly stimulated by TMPD plus ascorbate or by preincubation with ATP, and this stimulation was abolished by uncouplers. Oligomycin A, aurovertin, octylguanidine and atractyloside inhibited the stimulation by ATP, but slightly increased or did not affect the stimulation by TMPD plus ascorbate.</li> <li>The penetration of oxaloacetate and L-malate into liver mitochondria obeyed Michaelis–Menten Kinetics, and apparent K<sub>m</sub> values were respectively 40 μM and 130 μM at 20°. The K<sub>m</sub> values were similar both in the presence or absence of an energy-supply, but V<sub>max</sub> values were greatly increased by TMPD plus ascorbate or ATP.</li> <li>Arrhenius plots of the temperature-dependence of the penetration of oxaloacetate and L-malate above 12° gave activation-energies of +10 and +8 Kcal/mole respectively, both in the presence or absence of an energy-supply.</li> <li>The penetration of oxaloacetate and L-malate was competitively inhibited by D-malate, malonate, succinate, mesotartrate, maleate and citraconate, both in the presence or absence of an energy-supply. The K<sub>i</sub> values of these inhibitors were similar for penetration of both oxaloacetate and L-malate.</li> <li>Valinomycin or Gramicidin plus KCl had a twofold effect on the penetration of oxaloacetate in the presence of an energy-supply. In the presence of low concentrations of KCl, rates of penetration of oxaloacetate and L-malate were stimulated, but in the presence of higher concentrations of KCl rates of penetration were inhibited.</li> <li>EDTA and EGTA (up to 5 mM) had no effect on the rates of penetration of oxaloacetate into RLM prepared in the presence of 0.1 mM EDTA. If mitochondria were depleted of bound Ca<sup>2+</sup> by preparation in the presence of 1 mM EGTA, the stimulation of the penetration of oxaloacetate by TMPD plus ascorbate or by ATP was greatly reduced. The energy-dependence could be restored by addition of 0.1 mM CaCl<sub>2</sub>. It was concluded that bound Ca<sup>2+</sup> was required for the stimulation of oxaloacetate penetration by an energy-supply.</li> <li>Preliminary experiments have been performed to investigate the stoichiometry of the energy-requirement for the stimulation of oxaloacetate penetration into RLM. <em>Initial</em> rates of penetration of oxaloacetate were, measured spectrophotometrically, and 'extra' ATPase activity (see Klingenberg 1963), or oxidation of ascorbate was measured under identical conditions. It was found that 1.5–1.7 'extra' molecules of oxaloacetate entered the mitochondria per high-energy bond consumed, but as this only applies to the <em>initial</em> rate of penetration, it is not known whether the stoichiometry is fixed.</li> <li>These results suggest that oxaloacetate and L-malate enter mitochondria by a carrier-system which is probably the same for both these anions. The carrier system is energy-dependent and activated by the bound Ca<sup>2+</sup> of the mitochondria. Possible mechanisms for the carrier-system are discussed.</li> </ol> <p><strong>The Temperature-Dependence of Energy-linked Reactions in Mitochondria and Sub-mitochondrial Particles</strong></p> <ol type="1"> <li> The temperature dependence of the following energy-linked reactions were investigated:- <ol type="a"> <li>The energy-linked transhydrogenase in sub-mitochondrial particles from rat liver and beef heart.</li> <li>The ATP-driven reduction of NAD<sup>+</sup> by succinate in sub-mitochondrial particles from rat liver and beef heart.</li> <li>The stimulation of the respiration of HBHM by ADP.</li> <li>The stimulation of the penetration of oxaloacetate and L-malate into rat liver mitochondria by TMPD plus ascorbate or ATP.</li> </ol> </li> <li>Arrhenius plots of the temperature-dependence of all these reactions were similar in form. Plots of respiration-driven reactions did not show a marked break, but plots of ATP-driven reactions showed a sharp break in the temperature range 8–17°. Below this 'transition-temperature' the activation energies of the reactions were greatly increased.</li> <li>It is postulated that the sharp break in the Arrhenius plots of ATP-driven reactions reflect a configurational change in the enzyme(s) which catalyse the reversible formation of ATP in the terminal step of oxidative phosphorylation.</li> </ol>
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