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Title: A study of the cell envelope of Halobacterium salinarium
Author: Brown, Richard Henry
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1969
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The plasma membrane of Halobactertium, strain 1, an extremely halophilic bacterium, has been isolated and characterised. The cell envelope fraction (CEFB) was isolated from a cell homogenate by differential centrifugation. After dialysing the cell envelope fraction against distilled water and treating with nucleaaes, a fairly pure preparation of plasma membranes (NM) was obtained by centrifuging. The membrane -containing fraction was purified by gel filtration on Agarose, which yielded a purified membrane fraction (R) and a protein-nucleotide fraction (RP). A small molecular weight protein fraction P was separated from the purified membrane fraction either by gel filtration on Sephadex in a buffer containing phosphate and fluoride tons or by ultrafiltration. Protein fractions RUP1 and RUP2 were separated from the purified membrane by gel filtration on Agarose in the presence of 6M-urea. The remaining membrane- containing fraction, which was eluted in the void volume of the urea-Agarose gel, was coded RU. The organism was studied in batch culture; maximal growth was reached after 48 hours, after which time the cells were in the 'stationary phase.' The endogenous respiratory activity of the cells rose to a maximum at 40 hours and then declined steadily, but the viable count remained fairly steady. Analyses of the membrane fraction NM were made at various phases of growth up to a maximum of 160 hours. All the cell lipid was found to be concentrated in the membrane fraction. The amount of lipid expressed as a percentage of the salt-free dry weight of the cell remained constant, but both the total cell protein and the membrane protem fell durtng the period between 16 and 112 hours of growth. Also, the proportion of membrane to whole cell fell during this period. The menaquinone and the carotenoid pigment were localised exclusively in the membrane fraction. Both compounds exhibited maximal concentrations at 64 hours of cell growth and retained a constant molar ratio to each other regardless of the growth phase. The crude (NM) and purified (R) membrane fractions were both affected by magnesium ion. In the absence of the ion, the membrane disaggregates to small lipoprotein particles. Magnesium ions also assist in the binding of the amino sugar layer of the cell envelope and of the P and RP fractions to the membrane. The physico-chemica! properties of the fractions NM, R. RP, RUP and P have been investigated by a combination of amino acid analysts, gel electrophoresis, sucrose density gradient centrifugation and gel filtration on Sephadex or Agarose. In addition, the binding of magnesium ion to the membrane and the isotonic point of the membrane rractton R have been determined. The fraction R was found to be free from amino sugar and nucleotide. Fractions NM, R and RU contain all the cell lipid, and eytechreme, Fractions NM and R, and probably also fraction RU, contain the cell menaquinone and carotenoid. Evidence is presented that suggests that the fraction R contains the NADH oxidase and adenosine triphosphatase of the electron transport system. The action of the detergent sodium dodecyl sulphate (SOS) on the membrane fraction R was to break up the membrane into smaller particles. The disaggregation noccurred in two distinct steps. The disaggregated particles could be reaggregated to a fraction which resembled the original membrane by removing the SDS by dialysis or gel filtration. The disaggregated particles and also the reaggregated membrane fraction were subjected to gel filtration, gel electrophoresis and sucrose density gradient centrifugation, in order to determine whether or not the lipid and protein components of the membrane had been separated. A fraction which may be analogous to mitochondrial structural protein was isolated by ammonium sulphate fractionation of fraction R dissolved in a mixture of sodium deoxycholate, sodium chelate and SDS.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH301 Biology ; QR Microbiology