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Title: Studies on rat hepatic bile acid-binding proteins using photoaffinity labelling techniques
Author: Henderson, Colin J.
ISNI:       0000 0001 3552 6318
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1984
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The transcellular route that bile acids follow during the hepatic phase of the enterohepatic circulation has been a matter of controversy for some time. Among the modes of intracellular transport proposed have been free diffusion and partitioning within cellular organelles, vesicular transport, and association with cytosolic binding proteins. It is known that several groups of bile acid -binding proteins exist in rat hepatic cytosol; these include Z protein(s) and the glutathione S- transferases (Y proteins). A new group, the Y proteins, has been discovered recently and a purification scheme reported for two bile acid -binding proteins, which have been partially characterised. The aims of this study have been to investigate bile acid -binding proteins from rat hepatic cytosol, with particular reference to the Y proteins. The technique of photoaffinity labelling has been employed as a method of assessing the bile acid -binding potential of these proteins. The photoaffinity label used in these experiments has been [125I]- 3ßazidocholylhistamine, a photolabile derivative of cholic acid, radioiodinated to a specific radioactivity of 1900 Ci /mmol. This labelled molecule was shown to retain the properties of the parent bile acid with regard to hepatic transport. In photoaffinity labelling studies with cytosolic proteins from rat liver, previously undescribed bile acid - binding proteins have been identified in the Y' fraction. These proteins were purified using gel filtration, ion - exchange, chromatofocusing and hydroxyapatite chromatography. The resulting homogeneous proteins fell into two categories: - 1) Proteins (designated binders 5B, 6E and 7F) consisting of two identical subunits each having an approximate molecular mass of 15 000 - 19 000 Da. These showed a high specific incorporation of radioactivity after photoaffinity labelling, and were present at low concentrations. 2) Proteins (designated binders 5C, 5D and 8c) of molecular mass approximately 33 000 - 36 000 Da. These showed a lower specific incorporation of radioactivity after photoaffinity labelling than those in group 1, but had a higher total incorporation of [1251] owing to their higher concentration in the liver. These proteins were analysed by peptide "mapping" on reverse -phase high performance liquid chromatography to determine their genetic relationships. In addition, along with several glutathione S- transferases, they were photoaffinity labelled and peptide "mapped" to determine the position of the radioactive label, and thus the bile acid -binding site, within the molecule.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Rat bile proteins