Abstract
Changes in the Level of serum enzymes, notably 5'-nucleotidase and alkaline phosphatase, have frequently been used as a diagnostic aid for liver disease. Therefore the relationship between rat serum proteins and Liver plasma membrane was investigated using immunofluorescence and affinity chromatography. The extent of binding of the isolated plasma membrane to fluorescein-labelled anti-(rat serum) globulin fraction was examined, and a valid assay for proteins homologous to serum was developed. About 1% of the protein of the globulin fraction was capable of binding to the membrane. Washing the isolated membrane with 0.15 M-NaCl and 0.2 M-NaHCO[3] / pH 9. 0, resulted in the removal of the non-membraneous proteins as well as some of the genuine membrane proteins. The proportion of anti-(rat serum)-binding material washed off is about the same as the proportion of the total membrane proteins that is solubilised. However, when the binding of the isolated and salt-extracted membranes to anti-(liver plasma membrane) antiserum was examined, it was found that the washing procedure caused the exposure of a new binding site which was previously masked. Affinity chromatography of rat serum on immobilised anti-(plasma membrane) conjugate resulted in the isolation of serum proteins, about 4% of total serum protein, common to liver plasma membrane. Enzyme activity measurements showed that none of the 5'-nucleotidase and aIkaline nitro phenyIphosphatase was associated with the common proteins. However, the additional 5'-nucleotidase which appears in the serum of jaundiced rats was found to originate from the hepatocyte plasma membrane (Issa et al. , 1976). The microsomal fraction of rat liver homogenate was fractionated using a linear sucrose density gradient. The plasma membrane fragments recovered in the microsomal fraction were found to be heterogeneous in density. It was demonstrated that 5'-nucleotidase, the most commonly used plasma membrane marker, is concentrated in the material banding at a density of 1.14 g/ml while most of other plasma membrane markers were found at a density of 1.16 g/ml. The possibility that the light microsomal subfraction is derived largely from the blood-sinusoidal surface of the hepatocyte was confirmed by an in situ labelling with SITS. Examination of serum proteins after injection of L-[[14]C] fucose showed that the proteins common to liver plasma membrane and rat serum are labelled more slowly than other secretory proteins. In addition, plasma membrane fragments from the sinusoidal facing area of the liver cell seem to be labelled more rapidly than other plasma membrane fragments. The results provide evidence that the material reaching the sinusoidal part of the plasma membrane is either released into the blood directly or moves to other parts of the membrane, finally to the bile canalicular region.