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Title: The uptake of vitamin B12 by unicellular algae
Author: Borley, Mark Letchford
Awarding Body: University of London
Current Institution: Royal Holloway, University of London
Date of Award: 1987
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Vitamin B12 binding and uptake by two unicellular, marine algae, the autotrophic Dunaliella primolecta and the auxotrophic Pavlova lutheri, has been studied. Features of the extracellular binders found in the culture medium of micro-algae were examined to elucidate their role. The B12 binding capacity of these proteins was shown to be proportional to cell density, and they were relatively stable for at least one month. Nevertheless, cells bind the majority of vitamin until stationary phase. Membrane-bound and intracellular cytosolic binders have also been isolated, representing around 49 and 16 % respectively of the total B12 binding capacity of cultures, 35% typically being associated with the extracellular fraction. To discover the relationship between these proteins and the nature of the uptake process, characterisation was pursued. An affinity chromatography-based purification scheme was developed for the binders, and small quantities of these proteins were purified from all three phases. Gel filtration showed uniform molecular weights in excess of 500,000, and amino acid analyses revealed similar compositions. The identification of carbohydrate components was investigated using a lectin-binding assay. However, it was not found to be possible to determine sub-unit molecular weight by SDS gelelectrophoresis, and attempts at localisation of the cellular B12 binding sites using fluorescence microscopy and electron probe micro-analysis were unsuccessful. Interestingly though, similar vitaminbinding characteristics were demonstrated for isolatedchloroplasts and intact cells of D. primolecta. Aspects of B12 and its uptake by micro-algae arereviewed, and an explanation for extracellular B12 binding proteins involving non-specific release issuggested. A general model of two-stage uptake of the vitamin by micro-algae, with a primary stage of binding to a receptor protein in the plasma membrane and a secondary phase of energy-dependent internalisation, is also proposed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry