Binding of bile acids by 100 000g supernatants from rat liver

Abstract

1. The binding of glycocholic acid, chenodeoxycholic acid and lithocholic acid to rat liver 1000 000g supernatants was studied by equilibrium dialysis. 2. The binding characteristics of the bile acids suggest that the binding components are involved in bile acid transport. 3. When mixtures of [14C]lithocholic acid and liver supernatants were eluted from columns of Sephadex G-75, a prominent peak of [14C]lithocholic acid appeared with proteins of mol.wt. approx. 40000. A second, smaller, peak of [14C]lithocholic acid was eluted with proteins of mol.wt. approx. 100000. 4. The inclusion of cholic acid, glycocholic acid or chenodeoxycholic acid in the eluting buffer decreased the amount of [14C]lithocholic acid that was eluted with the higher-molecular-weight component.

A theoretical analysis of the use of zonal gel filtration in the detection and purification of protein-ligand complexes

“…about 40000 being gel-filtered through a Sephadex G-75 column of dimensions 2.6cm x 30cm (cf. Ockner & Manning, 1974;Strange et al, 1977). The sample contained 20nmol of protein in 2ml of buffer (e.g.…”

“…We have been using the technique to find out if tissues from certain teleosts contain proteins that. will bind long-chain fatty alcohols, so far with negative results (A. Bauermeister & J. R. Sargent, unpublished work), and we have also been puzzled by the fact that complexes with rather similar dissociation constants have markedly dissimilar capacities to survive gel filtration intact (Strange et al, 1977). We have therefore analysed a model of the process of zonal gel filtration, in an attempt to define what experimental conditions should be used and how tight the binding of protein to ligand has to be for the complex to emerge undissociated from the column.…”

“…Zonal gel filtration may be used to find out if a cell-free extract contains one or more proteins that will complex non-covalently with a given ligand. Several binding proteins have been discovered and partially purified in this way, such as ligandin (Levi et al, 1969;Ketterer et al, 1975), fatty acid-binding protein (Ockner & Manning, 1974), an oestrogen receptor (DeSombre et al, 1969) and two proteins that bind lithocholic acid (Strange et al, 1977); further examples may be found in the review by Wood & Cooper (1970). The technique is to gel-filter a mixture of extract and labelled ligand, and to assay the eluate for protein and label.…”

Binding of cholic acid to soluble proteins from rat liver

“…More recently the same authors [4] found two macromolecular species capable of binding lithocholic acid, one of mol. wt 40 000 and the other of mol.…”

“…In addition, intracellular proteins capable of binding bile acids have been described. Strange et al [3,4] have shown that bile acids bind to proteins present in high speed supernatant fractions from rat liver. Thus the transport of bile acids shows similarities to the transport of other organic anions.…”