The relation of bile proteins to serum and liver plasma membrane [proceedings].

Although there have been many studies on the low-molecular-weight constituents of mammalian bile, there is no clear agreement on the origin of bile proteins. An obvious source is the outer leaflet of the hepatocyte plasma membrane, and indeed it has been shown that bile is rich in enzymes typical of the plasma membrane (Holdsworth & Coleman, 1975; Evans et a/., 1976) and that bile salts are capable of solubilizing membrane proteins (Vyvoda et al., 1977). Moreover, it is clear that the 5’-nucleotidase of bile is immunologically identical with the enzyme present in liver plasma membrane (Evans et a/., 1976; Mullock P t al., 1977). However, immunological studies have also shown the presence of serum proteins, notably albumin and immunoglobulins, in human bile (Russel & Burnett, 1963). A protein with the immunological properties of serum albumin is also present in rat bile, but its electrophoretic mobility in sodium dodecyl sulphate-containing polyacrylamide gels differs from that of native serum albumin (Evans et a/., 1976). Further, the general pattern of rat bile proteins is quite distinct from that of serum proteins (Hinton & Mullock, 1977). Thus data available at present do not support a single source for bile proteins. It would seem clear that there is some solubilization of proteins, including enzymes such as 5’-nucleotidase, from the bile-canalicular face of hepatocytes, but that such solubilization cannot explain the presence of serum proteins in bile. Two hypotheses that would explain this observation are: (a) the ‘mistaken’ discharge of secretion granules containing serum proteins at the bile-canalicular rather than at the sinusoidal face of hepatocytes; (6) the discharge of serum proteins taken up and processed through the lysosomal system, for it has been suggested by deDuve & Wattiaux (1966) that lysosomes discharge their contents into the bile canaliculi. We have now attempted a systematic study of the immunological and electrophoretic properties of the proteins of rat bile with a view to improvingour understanding of their origin. Hooded rats of the University of Surrey strain were used in all experiments. Blood and bile were collected as described earlier (Mullock et al., 1977). Crossed immunoelectrophoresis using single or split antibody-containing gels was carried out as described by Axelsen e ta / . (1973). Anti-(rat serum) was from Mercia (Watford, Herts., U.K.). Anti(cholestatic rat serum) and anti-(liver plasma membrane) antisera were prepared by using the dosage schedule given by Mullock et al. (1977). Two dimensional electrophoresis with the first dimension run in agarose gel and the second dimension in a gradient polyacrylamide gel, was performed as described by Hinton & Mullock (1977). Crossed immunoelectrophoresis of rat bile proteins against anti-(rat serum) and anti(liver plasma membrane) revealed the presence of at least 14 proteins (Table 1). No difference was found between the reactions of bile with anti-(rat serum) and anti(cholestatic rat serum). Of the 14 proteins only one, a relatively minor component, reacted with anti-(liver plasma membrane) and not with anti-(rat serum). Three proteins reacted with anti-(rat serum) and with all preparations of anti-(liver plasma membrane) ; another protein reacted with anti-(rat serum) and with some anti-(liver plasma membrane) preparations. The relative strength of the reaction of all four proteins with the different antisera suggested that all were, in fact, serum proteins [our anti-(liver plasma membrane) antisera contain antibodies to a number of serum proteins (Issa et al., 1977)l. The remaining nine bile proteins reacted with anti-(rat serum), but not with anti-(liver plasma membrane). Bile lipoprotein (Manzato et al., 1976) did not appear to react with anti-(rat serum) or with anti-(liver plasma membrane), for none of the peaks obtained by crossed imrnunoelectrophoresis stained with Sudan Black B. Only nine proteins could be distinguished after two-dimensional electrophoresis of rat bile proteins. By measurement of the electrophoretic mobility in the first (agarose) dimension and by absorption of the bile with immobilized anti-(rat serum) it was possible to correlate most components identified on two-dimensional electrophoresis