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Complexes of rat alpha 1-macroglobulin and subtilisin are endocytosed by parenchymal liver cells.

ABSTRACT: Rat alpha 1-macroglobulin was isolated from plasma. Gel electrophoresis of the denatured and reduced protein showed two bands, with Mr values of 163 000 and 37 000. The large subunit contained an autolytic site. This subunit was also split after reaction of the macroglobulin with trypsin. Electron microscopy showed that the macroglobulin changed towards a more compact conformation after reaction with this proteinase. Subtilisin, or alpha 1-macroglobulin, was labelled with a sucrose-containing radio-iodinated group that stays in lysosomes after endocytosis and breakdown of the tagged protein. After intravenous injection into rats, alpha 1-macroglobulin was cleared from plasma with first-order kinetics, showing a half-life of about 9 h, whereas complexes of alpha 1-macroglobulin and subtilisin were cleared with half-lives of only 3 min. Liver contained about 60% of the label at 30 min after injection of complexes. About 90% of the liver radioactivity was found in parenchymal cells isolated after perfusion of the liver with a collagenase solution. Subcellular fractionation indicated a lysosomal localization of the complexes. We conclude that endocytosis by parenchymal liver cells is the major cause of the rapid clearance of alpha 1-macroglobulin-proteinase complexes from plasma.

SUBMITTER: Bergsma J

PROVIDER: S-EPMC1144679 | biostudies-other | 1985 Feb

REPOSITORIES: biostudies-other

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Project description:1. Receptor-mediated endocytosis of mannose-terminated glycoproteins in rat liver endothelial cells has been followed by means of subcellular fractionation and by immunocytochemical labelling of ultrathin cryosections after intravenous injection of ovalbumin. For subcellular-fractionation studies the ligand was labelled with 125-tyramine-cellobiose adduct, which leads to labelled degradation products being trapped intracellularly in the organelle where the degradation takes place. 2. Isopycnic centrifugation in sucrose gradients of a whole liver homogenate showed that the ligand is sequentially associated with three organelles with increasing buoyant densities. The ligand was, 1 min after injection, recovered in a light, slowly sedimenting vesicle and subsequently (6 min) in larger endosomes. After 24 min the ligand was recovered in dense organelles, where also acid-soluble degradation products accumulated. 3. Immunocytochemical labelling of ultrathin cryosections showed that the ligand appeared rapidly after internalization in coated vesicles and subsequently in two larger types of endosomes. In the 'early' endosomes (1 min after injection) the labelling was seen closely associated with the membrane of the vesicle; after 6 min the ligand was evenly distributed in the lumen. At 24 min after injection the ligand was found in the lysosomes. 4. A bimodal distribution of endothelial cell lysosomes with different buoyant densities was revealed by centrifugation in iso-osmotic Nycodenz gradients, suggesting that two types of lysosomes are involved in the degradation of mannose-terminated glycoproteins in liver endothelial cells. Two populations of lysosomes were also revealed by sucrose-density-gradient centrifugation after injection of large amounts of yeast invertase. 5. In conclusion, ovalbumin is transferred rapidly through three endosomal compartments before delivering to the lysosomes. The degradation seems to take place in two populations of lysosomes.

Project description:Periportal and perivenous parenchymal cells were isolated by the digitonin-pulse perfusion method. The digitonin-pulse perfusion was shown to lead to selective lysis of the correct zone with a straight and sharp border of two to three cells. The mean ratios of alanine aminotransferase activity (a marker for periportal parenchymal cells) and glutamine synthetase activity (a perivenous marker) of periportal to perivenous parenchymal cells were 1.76 and 0.025 respectively. Cells were incubated in vitro with 125I-asialo-orosomucoid (ASOR), 125I-trypsin-activated alpha 2-macroglobulin (alpha 2M-T) or 125I-beta-migrating very-low-density lipoprotein (beta-VLDL), in order to determine the zonal distribution of the asialoglycoprotein receptor (ASGPr), the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein (alpha 2Mr/LRP) and the lipoprotein-remnant receptor, respectively. Maximum binding capacity for 125I-ASOR on parenchymal cells showed a periportal/perivenous ratio of 0.70. The periportal/perivenous ratio of Bmax. values of binding of 125I-alpha 2M-T to parenchymal cells was 1.51. The Bmax. values of binding of 125I-beta-VLDL, however, were about equal for both cell populations. It is concluded that the maximum binding capacity of the ASGPr on isolated periportal parenchymal cells is 0.70 times that of perivenous parenchymal cells. The 1.51-fold higher expression of the alpha 2Mr/LRP on periportal cells, compared with perivenous parenchymal cells, indicates a zonal specialization for the uptake of the suggested multiple ligands. In contrast, the observed homogeneous distribution of the lipoprotein-remnant receptor is in accordance with the suggestion that lipoprotein remnants bind to a specific receptor, which is different from the alpha 2Mr/LRP. The zonal heterogeneity in the expression of receptors suggests that receptor-dependent uptake pathways are under zonal control, leading to intrahepatic heterogeneity in the removal of ligands from the blood circulation.

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Complexes of rat alpha 1-macroglobulin and subtilisin are endocytosed by parenchymal liver cells.

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