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Title: Studies on some aspects of oxidative phosphorylation
Author: Haslam, J. M.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1967
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This thesis presents the results of studies on some aspects of oxidative phosphorylation, and of mitochondrial reactions which utilise the energy of oxidative phosphorylation. The Stoichiometry of Oxidative Phosphorylation

  1. 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 P1 esterification.
  2. 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.
  3. The high P:O ratios reported by Smith andamp; Hansen (1964), Gurban andamp; Cristea (1964, 1965) and Lynn andamp; Brown (1965) were shown to be caused by an underestimation of oxygen consumption.

The Primary Phosphate Acceptor in Oxidative Phosphorylation

  1. The nature of the nucleotide acceptor, to which P1 is first linked in oxidative phosphorylation, was investigated by measuring the labelling by 32P1 of the bound adenine nucleotides of HBHM and phosphorylating submitochondrial particles (ETPH) during the first 2 min. of succinate oxidation.
  2. In all labelling experiments in the absence of uncouplers AT32P had a higher specific radioactivity than AD32P, and it was concluded that ADP is the primary endogenous acceptor of 32P1. This disagrees with the results of Ozawa andamp; MacLennan (1965), who claimed that AD32P contained most of the radioactivity in such experiments. Possible reasons for this discrepancy are discussed.
  3. In the presence of uncouplers, AD32P had a higher specific activity than AT32P in labelling experiments with HBHM. The labelling of AD32P 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 GT32P → AD32P → AT32P, catalysed by succinate thiokinase, nucleoside monophosphate kinase and myokinase, as shown by Heldt andamp; Schwalbach (1967) for rat liver mitochondria.
  4. 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 Mg2+. In the absence of Mg2+, or if a concentration of EDTA greater than that of Mg2+ 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.
  5. It was concluded that ADP is the primary external phosphate acceptor in HBHM, and that AMP is only indirectly phosphorylated in oxidative phosphorylation via the myokinase reaction, which requires added Mg2+. 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.

The Purification of Coupling Factors, and the Effect of NAD+ and NADH on the AD32P-ATP and 32Pi-ATP Exchange Reactions Catalysed by such Fractions

  1. 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% (NH4)2SO4 fraction of the supernatant from sonicated HBHM increased the P:O ratios of 'salt-extracted particles' (Green et al., 1963) and METPH (Linnane andamp; Titchener, 1960) with NADH or succinate as substrate.
  2. This crude preparation of coupling factor had a high AD32P-ATP exchange activity, and a small 32Pi-ATP exchange activity which depended in a complex way on the relative concentrations of Pi, ADP and ATP, and was inhibited by uncouplers or inhibitors of oxidative phosphorylation.
  3. The effect of NAD+ and NADH on the AD32P-ATP and 32Pi-ATP exchange reactions catalysed by the 0-45% (NH4)2SO4 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.
  4. The Pi-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 Pi-ATP exchange activity is associated with small fragments of the mitochondrial membrane and is not a soluble or homogenous enzyme.
  5. It was shown that although much of the AD32P-ATP exchange activity of the crude coupling factor was associated with myokinase, there was an excess of AD32P-ATP exchange activity which was possibly associated with the coupling factor.

The Mechanism of Penetration of Oxaloacetate and L-malate into Mitochondria

  1. 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 mandmu;.
  2. 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+ was a measure of the penetration of L-malate.
  3. 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.
  4. The penetration of oxaloacetate and L-malate into liver mitochondria obeyed Michaelis-Menten Kinetics, and apparent Km values were respectively 40 andmu;M and 130 andmu;M at 20°. The Km values were similar both in the presence or absence of an energy-supply, but Vmax values were greatly increased by TMPD plus ascorbate or ATP.
  5. 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.
  6. 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available