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Title: A study of the cell membrane and enzymes of Halobacterium salinarium
Author: Bellingham, Francis
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1972
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1. A colourless mutant, strain 1M, of the extreme halophile Halobacterium salinarium strain 1 was shown to be more sensitive to changes of NaCl concentration than the wild-type. The levels and cellular distribution of several enzymes were determined in each strain and the sensitivity to changes of NaCl investigated for selected membrane bound and soluble enzymes. ATPase, acid phosphatase, alkaline phosphatase and glycerol dehydrogenase of both strains all showed typical halophilic enzyme responses with optimum activity only in the presence of 2-5 - 4·0M salt. 2. Malate dehydrogenase from strain 1 was extracted and purified. The purified preparation showed an 850 fold increase of specific activity Over the crude extract. The fluorescence probe 1- 8 AN was used to investigate possible conformational changes induced in the purified enzyme by variations of NaCl concentration. Results indicate that upon removal of salt ANS fluorescence is enhanced possibly implying that unfolding of the protein occurs. 3. The cell membrane of strain 1 and 1M has been prepared from cell envelopes and purified by gel- filtration on Agarose Some Purification resulted in the release of a low M. W. ( "V 2500) polar protein which contained some nucleotide material. Electron micrographs of crude and purified membrane showed abundant triple layered structures and vesicles typical of classical unit membrane structures. Urea caused the release of highly polar fractions which were isolated by PGE and characterised by gel-filtration and amino acid analysis. 4. The membrane lipid of strain 1 and 1M were extracted and a preliminary characterisation carried out. sides the lack of carotenoid in strain 1M other differences in the lipid composition were found between the two strains. train 1M contained less menaquinone and at least two different phospho-lipids. Lipid-free membrane protein was extracted by a method combining organic solvent extraction and gel- filtration. The membrane protein of both strains was shown to be a polar in character and heterogeneous by separation on PGE. A structural protein fraction which may b similar to SP obtained from other membrane sources was isolated from purified membrane. 5. Purified membrane was solubilised with mixed anionic detergents (sodium dodecyl sulphate, sodium cholate and sodium deoxycholate ) and separated by gel- filtration into protein-rich and lipid- rich fractions. The lipids from each of these fractions were extracted and compared with total mernbrane extracts. The lipid component of the protein-rich fraction appeared to contain mainly polar lipids. 6. The protein-rich and lipid-rich fractions were reaggregated in the presence of 10~ Mg2+ or 5mM spermine to form reconstituted membrane . Reconstituted membrane appeared in e. m. s to contain unit membrane structures . Like native membrane, reconstituted membrane could only be disrupted by anionic detergentst indicating that hydrophobic bonding was of prime importance in the interaction between protein and lipid. Experiments in which membrane protein was labelled with the florescent probe DNS indicated that the membrane protein and lipid fractions were stabilised to some extent when they interacted. However, significant differences were observed in the behaviour of dansylated native membrane and dansylated reassembled membrane indicating that the organisation of protein and lipid in each may not be directly comparable.
Supervisor: Not available Sponsor: Science Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry