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
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
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  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.