Galactose‐Specific Endocytosis in Rat Liver

Abstract

Galactosylated bovine serum albumin (galBSA) and its peroxidase‐conjugate (galBSA‐HRP) are rapidly cleared after intravenous injection and accumulate in rat liver. Competition experiments indicate that these galBSA derivatives are taken up by the galactose‐specific receptor. By cytochemistry on liver slices, galBSA‐HRP is found almost exclusively in hepatocytes, in similar structures as those reported for asialoorosomucoid. After differential centrifugation of liver homogenates, galactosylated ligands are concentrated in the light mitochondrial (L) and the microsomal (P) fractions. Together, these two fractions contain roughly 80% of the galBSA or of its peroxidase‐conjugate present in the homogenate 10 min after injection to the animals. By isopycnic centrifugation of L or combined LP fractions in sucrose gradients, ligand‐containing structures equilibrate at low densities (1.11–1.13 g/ml). Up to 52‐fold enrichment over the homogenate can be achieved for these structures, with a 14% yield. By isopycnic centrifugation, the distribution of ligand‐containing structures is clearly distinct from the bulk of plasma membrane (5′‐nucleotidase), endoplasmic reticulum (glucose‐6‐phosphatase) and lysosomes (cathepsin B), but considerable overlapping with galactosyltransferase, a marker for the Golgi complex, is observed. GalBSA‐HRP‐containing structures have been identified by ultrastructural cytochemistry. In low density fractions, labelled structures are vesicles or tubules heterogenous in size and shape and surrounded by a smooth membrane. Other components of such preparations are mostly Golgi and endoplasmic reticulum elements. Quantitative assessment of the purity in our best preparations of ligand‐containing structures leads to the inference that they are about 50% pure. It may nevertheless be concluded that they constitute a biochemically and morphologically distinct intracellular compartment. This compartment could be specialized in ligand‐receptor and ligand‐ligand sorting.

Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles.

“…z LP fractions (Sml) were layered on the top of a 32-m1 linear sucrose gradient (from 1 .1 to 1 .3 g/ml in density), buffered with 3 mM imidazole/HCI, pH 7 .0. After centrifugation for 24 x 106 g~min in a VTi 50 rotor (Beckman Instruments), l4 fractions were collected and analyzed as previously described (23). The two fractions containing the highest radioactivity (average density : I .12 g/ml) were pooled and will be further referred to as LP, pool.…”

“…Ultrathin sections were examined unstained under 60 kV in a EM 301 Philips electron microscope . (23). For DAB cytochemistry, the LP, pool corresponding to the peak of the [3H]-ga1BSA-HRP distribution (density, 1.11-1 .13 g/ml) was incubated at 25°C in buffered sucrose (density, 1 .1 g/ml), containing 5.5 mM DAB and 11 mM HZOz.…”

“…In comparison, the DAB-induced density shift takes advantage of a highly sensitive, exogenous enzyme, that can be introduced into specific organelles along the endocytic pathway by fluid-phase or receptor-mediated endocytosis (7,23,27,29,33). In addition, this procedure is simple, fairly reproducible, and proved useful to analyze and to purify ligand-HRP-containing organelles (24).…”

Receptor Cell Biology: Receptor-Mediated Endocytosis

“…Endosomes are tubulovesicular structures of various sizes and shapes (17,42,47). Upon endocytosis a ligand initially encounters endosomes with relatively small vesicular volumes.…”

Receptor-mediated endocytosis of polymeric IgA and galactosylated serum albumin in rat liver. Evidence for intracellular ligand sorting and identification of distinct endosomal compartments

“…All sucrose solutions were buffered with 3 mM imidazole at pH 7.0. Electron microscopy was performed as described in [7] except that the 3,3'-diaminobenzidine solution was adjusted to pH 6 instead of 7.…”