Browsing by Author "Pitt, Michael Arthur"

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Studies on the Role of Phospholipid, Vitamin E and Selenium in Microsomal Electron Transport
(Te Herenga Waka—Victoria University of Wellington, 1973) Pitt, Michael Arthur
The lipid requirement of liver microsomal electron transport enzymes has been studied using agueous acetone extractions. The liver microsomes were extracted with acetone:water 100:4 (v/v), 100:7 (v/v) and 90:10 (v/v). The extractions removed 24, 41 and 59% of the organic phosphorus respectively. The removal of 24 or 41% of the organic phosphorus did not cause loss of NADH-cytochrome c reductase activity. However removal of 59% of the organic phosphorus resulted in a substantial reduction of NADH-cytochrome c reductase activity. This enzyme system is known to have a lipid requirement, but the studies undertaken for this thesis indicate that this lipid requirement is not manifest unless more than 41% of the organic phosphorus is removed. The aqueous acetone extractions described above did not affect the activities of either NADH-ferricyanide reductase or NADPH-cytochrome c reductase. All of the above aqueous acetone extractions resulted in very large reductions in the activity of NADPH-dependent lipid peroxidation. This enzyme system utilizes microsomal phospholipid as substrate and the removal of phospholipid by aqueous acetone extraction would be expected to lower the activity of the enzyme system. The cytochrome b5 content of liver microsomes was slightly increased by aqueous acetone extraction. The effect of prior lipid peroxidation on microsomal electron transport enzymes has been investigated. NADH-cytochrome c reductase, NADH-ferricyanide reductase, NADPH-cytochrome c reductase and NADPH-dependent lipid peroxidation activities were not affected by prior lipid peroxidation. However NADPH-dependent aniline hydroxylation activity was diminished by prior lipid peroxidation. The studies outlined in the above paragraphs were carried out using sheep liver microsomes. The characteristics of electron transport in sheep liver microsomes, which are as yet unreported, are given in this thesis. The effect of vitamin E on the electron transport components of rat liver microsomes was studied. Vitamin E was observed to completely inhibit NADPH-dependent and non-enzymic lipid peroxidation when added by diet (600mg of vitamin E/kg of diet), or when added to microsomes. The vitamin was without effect on other microsomal electron transport enzymes. A deficiency of vitamin E induced by diet did not lead to increased liver microsomal NADPH-dependent lipid peroxidation activity. Vitamin E deficiency did not affect the activities of other microsomal electron transport components. The inhibition of NADPH-dependent lipid peroxidation activity by dietary vitamin E could be reversed by removing the vitamin E from the diet or by extracting microsomes with acetone:water 100:4 (v/v). A 50% level of inhibition of NADPH-dependent lipid peroxidation activity in microsomes was observed at a concentration of 7 μg of dl-alpha-tocopheryl acetate per mg of protein and at 14 μg of d-gamma-tocopherol per mg of protein. The data obtained on the effect of vitamin E on microsomal electron transport is consistent with the concept that vitamin E is associated in a non-covalent manner with the unsaturated fatty acids of microsomal phospholipids. The effect of dietary induced vitamin E and selenium deficiency, and selenium deficiency on microsomal electron transport in rat liver was investigated. Neither vitamin E and selenium deficiency nor selenium deficiency affected the activities of the microsomal electron transport components. Titration of the microsomal sulphydryl groups with p-chloromercuribenzoate inhibited enzymic lipid peroxidation activity but resulted in increased non-enzymic lipid peroxidation. It appears that sulphydryl groups are able to control non-enzymic lipid peroxidation. NADH-cytochrome c reductase, NADH-ferricyanide reductase and NADPH-cytochrome c reductase activities were solubilized from the microsomal membrane by triton X-100 extraction. This extraction also solubilized cytochrome b5 and phospholipid. Only low NADPH-dependent lipid peroxidation activity was released from the microsomes by triton X-100.
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The synthesis of 8-cyano-7, 8-dihydro-7-oxobenzo [4,5] cyclo-hepta [1,2,3-de] naphthalene
(Te Herenga Waka—Victoria University of Wellington, 1969) Pitt, Michael Arthur
In 1952 Pullman et al. Pullman, B., Pullman, A., Berthier, G., and Pontis, J., J. Chim. Phys., 1952, 49, 20. made a theoretical study of pleiadiene (I), acepleiadylene (II), pleiadene (III) and benzo[4,5] cyclohepta- [1,2,3-de] naphthalene (IV). Boekelheide and Vick Boekelheide, V., and Vick, G. K., J. Am. Chem. Soc., 1956, 78, 653 were able to synthesize pleiadiene (I) and acepleiadylene (II) and it was found that their physical and chemical properties agreed with those calculated by Pullman et al. Cava and Schlessinger, Cava, M. P., and Schlessinger, R. H., J. Am. Chem, Soc., 1963, 85, 835 in 1963, attempted to synthesize pleiadene (III) and its acenaphthylene analogue (v). They showed these were much less stable than pleiadiene (I) or acepleiadylene (II). Pleiadene (III) and its acenaphthylene analogue (v) could only be isolated as a dimer resulting from a Diels-Alder self condensation. This instability might be expected as acenes decrease in stability as the number of linearly fused rings increases.