Bile Salt Binding to Serum Components. Taurocholate Incorporation into High-Density Lipoprotein Revealed by Photoaffinity Labelling

Abstract

1. Photoaffinity labelling of human serum albumin with the sodium salts of (3fl-azido-7a,12~-dihydroxy-5~-cholan-24-oyl)-2-amino[2-3H(N)]ethanesulfonic acid, (7,7-azo-3~,12ct-dihydroxy-5P-cholan-24-0yl)-2-amino[2-~H (N)]ethanesulfonic acid and (1 1 [-azido-12-0~0-3a,7a-dihydroxy-5~-cholan-24-oyI)-2-amin0[2-~H (N)]ethanesulfonic acid resulted, in each case, in a considerable covalent incorporation of radioactivity into the protein.2. Photoaffinity labelling of whole serum, obtained from fasting test persons, revealed with all three photolabile derivatives of taurocholate at the physiological concentration of 2.1 pM the incorporation of radioactivity not only into albumin but also into high-density lipoprotein, as demonstrated by density gradient centrifugation and by immunological characterization.3. The bulk of radioactivity incorporated into high-density lipoprotein by photoaffinity labelling of whole serum was found to have been associated with the lipids. Only l0-200/, of the label was covalently bound to apolipoproteins, predominantly to the apolipoproteins A-I and A-11.4. The interaction of taurocholate with high-density lipoprotein has been confirmed by density gradient centrifugation using I4C-labelled taurocholate. It is assumed that the interaction of taurocholate with high-density lipoprotein is physiologically of significance.

Bile‐Salt‐Binding Polypeptides in Plasma Membranes of Hepatocytes Revealed by Photoaffinity Labelling

“…Under physiological conditions, the concentration of total bile salt is lower than 10 pM in peripheral blood of rats [53] and several-fold higher in portal blood [54,55]. However, due to the binding of bile salts to albumin [30,56] and their incorporation into high-density lipoprotein [30] the actual concentration of free bile salts is far lower. In order to identify, on the one hand, all polypeptides of the membrane subfractions which bind bile salts and to minimize, on the other hand, the unspecific interactions with the lipid part of the cell membrane, the photolabile derivatives were applied in the concentration range of 2 pM and the mem- (B) Sodium salt of (7,7-azo-3a,12a-dihydroxy-5~-cholan-24-oyl)-2-aminoethanesulfonic acid.…”

“…It is well known that albumin binds unconjugated and conjugated bile salts [56,60,61] and exerts a specific binding function in bile salt transport in blood [30,51]. It may interact with specific receptors at the surface of hepatocytes [62-641, and it has been proposed that albumin, in some way or other, is involved in the uptake process for bile salts by hepatocytes [65].…”

“…Photoaffinity labelling was performed using the conditions and the experimental setup as described [30]. Of the different buffers which can, in principle, be used for photoaffinity labelling of membrane subfractions we chose to use 30 -50 mM sodium phosphate buffers, pH 7.1, because sodium phosphate buffer proved least critical in subsequent investigations of other organelles [52] and of isolated hepatocytes (Buscher, Fricker, Gerok, and Kurz, unpublished) by photoaffinity labelling.…”

“…Photolyses were carried out at 30 "C in cylindrical cuvettes, made of Duran glass, using the apparatus and the technique described [30].…”

“…Therefore we have used the different photolabile derivatives of conjugated bile salts (Fig. 2) which have proved their effectivity in the identification of bile-salt-binding polypeptides in serum [30,51].…”