Electron transport systems exist in the plasma membranes of all cells. Although not well characterised they play roles in cell growth and proliferation, hormone responses and other cell signalling events, but perhaps their most important role, especially in erythrocytes, is enabling the cell to respond to changes in both intra- and extracellular redox environments. Human erythrocytes possess a transmembrane electron transport capability that mediates the transfer of reducing equivalents from reduced intracellular species to oxidised extracellular species and is concomitant with proton extrusion. In the work for this thesis I showed that erythrocyte membranes contain a transmembrane WST-1 (water soluble tetrazolium-1) reductase activity that uses reducing equivalents from intracellular NADH to reduce extracellular WST-1. The rate of WST-1 reduction was increased by the presence of phenazine methosulfate and, although of low activity, it showed similar properties to a previously reported transmembrane NADH-oxidase activity. 1H NMR experiments showed that WST-1 was reversibly bound to the membrane and/or proteins in the membrane within the timeframe of the NMR experiment, confirming the location of the WST-1 reduction. Preliminary attempts to purify NADH:WST-1 reductase and NADH:ferricyanide reductase activities from the erythrocyte plasma membrane were inconclusive. The protein(s) responsible for the reduction of these oxidants appear to be of low abundance in the plasma membrane and may be part of a larger protein complex. Further work on the isolation of these redox activities is required before the protein(s) involved can be identified with any confidence. The ability of cells to export electrons suggests that an electron import mechanism might also exist to re-establish the cell�s redox-buffering equilibrium under conditions of oxidative stress. This hypothesis was tested in glucose-deprived erythrocytes using reduced glutathione and NADH as extracellular electron donors. It was shown that neither reduced glutathione nor NADH donated reducing equivalents through a transmembrane redox system. Extracellular NADH was, however, able to produce profound changes in starvation metabolism and methaemoglobin reduction rates. The addition of extracellular NADH caused a six-fold increase in the rate of lactate production above that observed in glucose-starved controls, together with a concomitant decrease in pyruvate production. In erythrocytes containing high levels of methaemoglobin, extracellular NADH increased the rate of methaemoglobin reduction in both the presence and absence of glucose. These results were explained by the leakage of lactate dehydrogenase from erythrocytes due to an admittedly low level of haemolysis. This caused the displacement of the intracellular pseudo-equilibrium of the lactate dehydrogenase reaction via transmembrane exchange of lactate, allowing the conversion of extracellular pyruvate to lactate and resulted in an increase in intracellular NADH concentrations. The latter increased the rate of methaemoglobin reduction. In conclusion, the work described in this thesis showed that erythrocyte membranes do not contain mechanisms for importing electrons or reducing equivalents from extracellular reduced glutathione or NADH. Erythrocytes do, however, contain an electron export system which can reduce extracellular oxidants such as WST-1 and the activity of this system depends on an intricate balance between intracellular antioxidants and enzyme activities. There is much still to be learnt about plasma membrane redox systems, little is known, for example, about the protein composition, mechanism of action, and the in vivo conditions under which these systems are most active.

1. The work described in this thesis forms part of an investigation of eukaryotic gene control. The system studied was the avian erythroid cell series since it is possible to isolate pure populations of the various cell types which have well-defined biochemical activities. These cells contain an unusual tissue - specific histone H5, which may be involved in the progressive repression of transcription observed as these cells differentiate. Although the gene controlling function of this histone must be at a very gross level, this represents a unique opportunity to investigate one facet of gene control. Probably the most sensitive technique is to assay for specific messenger RNA and gene sequences by hybridisation to an appropriate probe. The aim of this thesis was to prepare such a probe from H5 mRNA and to use it to calculate the reiteration frequency of the H5 gene in the chicken genome. 2. The cells employed were chicken reticulocytes since the only histone made in these cells is H5. Experiments were conducted which demonstrated that H5 mRNA is probably a minor species compared to globin mRNA in these cells. Furthermore, calculations indicate that the two mRNAs are probably of similar molecular weight which may complicate the isolation of H5 mRNA. As a result globin mRNA was first purified and characterised. Properties which may have proved useful in the separation of this mRNA from H5 mRNA are discussed. The globin mRNA was used to optimise techniques for the in vitro translation and identification of chicken mRNAs. This was considered necessary as mRNAs from different sources vary in the conditions required for optimal translation and it was reasoned that mRNAs from the same cell would have similar optima. 3. Total polysomal RNA was fractionated on the basis of size and poly A content. Although large amounts of globin mRNA were present, H5 mRNA could only be detected in the non - poly A containing RNA. Even in this fraction however, there was still a large excess of globin mRNA which was difficult to remove due to the demonstrated similarity of their molecular weights. 4. Since it had proved impossible to isolate the H5 mRNA by conventional techniques, immunological methods of isolating the polysomes producing H5 were investigated. Using immunoadsorbents, mRNA was prepared in small amounts which programmed the synthesis in vitro of more than 70 % H5. The yield and specificity were improved by modifying the procedure to indirect immunoprecipitation followed by oligo ( dT ) - cellulose chromatography. The resulting mRNA programmes the synthesis in vitro of more than 90 % H5. The chemical purity of the mRNA is discussed. 5. The immunologically prepared H5 mRNA was not copied into cDNA by RNA - dependent DNA - polymerase. Since this was probably due to the lack of a 3 ' poly A tract on the mRNA, an enzyme was purified and characterised which would add such a tract. The enzymically modified mRNA could then be copied into cDNA of high specific activity. 6. The H5 cDNA was characterised in terms of size and fidelity of copying. By hybridisation analysis it was demonstrated that the amount of contaminating rRNA and globin mRNA complementary sequences present in the cDNA was insignificant. The complexity of the cDNA was shown to be of the same size as the H5 mRNA and will back hybridise to this mRNA to greater than 75 %. These results are discussed to demonstrate that the cDNA is a faithful copy of H5 mRNA. The possible uses of the resulting probe are also discussed. 7. The H5 cDNA was employed to quantify the number of H5 genes in the chicken genome. The significance of this result is discussed in terms of the known reiteration and organisation of histone genes in other species, and the possible role of H5 as a gene control agent. / Thesis (Ph.D.)--Department of Biochemistry, 1975.

Thesis (Ph.D)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmaceutics. Title from title-page of electronic thesis. Bibliography: leaves 249-255.

Thesis (M. Phil.)--University of Hong Kong, 1984.

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published_or_final_version / Biochemistry / Master / Master of Philosophy

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