Galactosylated BSA (gaIBSA) and its conjugate to horseradish peroxidase (gaIBSA-HRP) enter the galactose-specific pathway of hepatocytes . 10 min after intravenous injection, structures containing either ligand sediment mostly between 33,000 and 3 x 106 g~min (LP fraction) and have an equilibrium density of 1 .11-1 .13 g/ml in sucrose gradients (Quintart, J., P. J . Courtoy, J . N . Limet, and P. Baudhuin, 1983, Eur . J. Biochem ., 131 :105-112) .Such low density fractions, prepared from rats given gaIBSA-HRP, were incubated for 30 min at 25°C in 5.5 mM 3,3'-diaminobenzidine (DAB) and 11 mM H202 in buffered sucrose . Upon equilibration in a second sucrose gradient, the gaIBSA-HRP distribution shifted towards higher 01 .19 g/ml) density, but the bulk of protein remained at low density . In the absence of H202, gaIBSA-HRP distribution was also found at low density . As observed by electron microscopy, particles equilibrating at higher density after DAB cytochemistry were largely made of vesicles or tubules filled with DAB reaction product. The density shift of gaIBSA-HRP-containing organelles after incubation with DAB and H zOz is attributed to the trapping of HRP-oxidized DAB inside the host organelles .If the low density fractions isolated from a rat injected with [3 H]gaIBSA-HRP were mixed in vitro with similar fractions from another rat given [' 4C]gaIBSA, the 3 H distribution shifted after DAB cytochemistry, but the '4C distribution was essentially unaffected . By contrast, if both derivatives were injected simultaneously, a concomitant density shift was observed .In conclusion, the DAB-induced density shift was specific to ligand-HRP-containing organelles . The potentials of the method include the purification of HRP-containing particles and the study of their association to ligands, fluid-phase tracers, or marker enzymes .After receptor-mediated internalization (3, 28), ligands are rapidly transferred to an "intermediate compartment" (34,37), often referred to as "endosomes" (14) or "receptosomes" (37). To assess the purity of subcellular fractions enriched in ligand-containing organelles, we recently used ligands conjugated to horseradish peroxidase (HRP),' an approach that proved successful in intact liver (29,33). Glutaraldehydefixed fractions were incubated in 3,3'-diaminobenzidine ' Abbreviations used in this paper: DAB, 3,3'-diaminobenzidine; gaIBSA, galactosylated BSA; gaIBSA-HRP, gaIBSA conjugated to horseradish peroxidase (HRP) .(DAB) and HZOz (12) and processed for electron microscopy . The organelles containing ligand-HRP were identified by the DAB reaction product (23). We report here that the DAB procedure can also be applied to unfixed subcellular fractions. Moreover, the accumulation of polymerized DAB inside HRP-containing organelles induces a major increase in their equilibrium density, while other organelles of the same fraction are essentially unaftècted.It has already been reported that cytochemical procedures may be applied to the isolation of specific organelles with endogeno...

Rat liver organelles involved in receptor-mediated endocytosis were labeled with a conjugate of galactosylated BSA to horseradish peroxidase ([3H]gaIBSA-HRP), injected 10 min before sacrifice . These organelles were recovered at low density (1 .11-1 .13 g/ml) in sucrose gradients (Quintart, J ., P. J. Courtoy, J. N . Limet, and P. Baudhuin, 1983, Eur. J. Biochem ., 131 :105-112) . Upon incubation of such low density fractions in 3,3'-diaminobenzidine (DAB) and H 202 and equilibration in a second sucrose gradient, gaIBSA-HRP-containing particles selectively shifted towards heavier densities (Courtoy, P. J ., J . Quintart, and P. Baudhuin, 1984, J. Cell Biol., 98:870-876, companion paper), resulting in up to 250-to 300-fold purification with respect to the homogenate. The most purified preparations, wherein DAB-stained structures represented -85% of the total volume of particles, contained only trace activities of enzymes usually regarded as markers for other subcellular entities . These minor activities could reflect either contamination or true enzyme association to the ligandcontaining structures . Considering the latter hypothesis, at most 1 .0% of alkaline phosphodiesterase I and 2 .6% of 5'-nucleotidase (markers for plasma membrane), 3 .6% of N-acetyl-ß-glucosaminidase (lysosomes), and 6 .0% of galactosyltransferase (Golgi complex) from the homogenate would be associated with the whole population of ligand-containing organelles . After DAB cytochemistry on liver fixed 10 min after gaIBSA-HRP injection, ligand-containing structures accounted for 0 .78-0.89% of the fractional volume of the hepatocytes and displayed a membrane area of 2,100 cm2 /Cm 3, compared with 6,700 Cm2/Cm 3 for the pericellular membrane . Altogether, our data support the hypothesis that these ligand-containing organelles are structurally distinct from plasma membrane, lysosomes, and Golgi complex .Rapidly after interiorization, numerous ligands taken up by receptor-mediated endocytosis (7,31,44) are found associated with structures equilibrating at low density (15, 21-23, 26, 30, 35, 40, 41, 48). Electron microscopy shows that ligands are concentrated in clathrin-coated pits or vesicles, and rapidly transferred into electron-lucent, smooth surfaced organelles (35,45,47

Rat polymeric IgA (pIgA) and galactosylated bovine serum albumin (GalBSA), once injected to rats, are avidly taken up by hepatocytes via receptor‐mediated endocytosis. Of injected pIgA, 64% was transferred undigested into bile within 3 h, with a peak at 30–45 min. GalBSA was essentially digested in lysosomes. By electron microscopy using ligand‐peroxidase conjugates, both ligands were internalized through coated pits/coated vesicles into similar electron‐lucent vesicles and tubules. Subsequently, pIgA remained mostly associated with small vesicles clustering around and fusing with bile canaliculi, while GalBSA was predominantly found in large, heterogeneous endocytic structures and in lysosomes. By subcellular fractionation, they were associated at 3 min after injection with structures that similarly sedimented in the P fraction (250000−3 · 106×g×min) and equilibrated at densities of about 1.13 g/ml in sucrose gradients. At 10 min and 20 min, pIgA distribution remained mostly in the P fraction at the same equilibrium density. A minor component of the pIgA distribution was found at the density of lysosomes, but contrary to lysosomal enzymes, its distribution was not affected by Triton WR 1339. In contrast to pIgA, GalBSA was progressively recovered in the L fraction (33000−250 000 × g × min) with organelles equilibrating around 1.11 g/ml, and, by 20–45 min, was found in the ML fraction (10 000−250 000 × g × min), around 1.20 g/ml, i. e. in lysosomes. Chloroquine did not reduce the efficiency but delayed the secretion of pIgA into bile. Similarly, it did not affect the uptake of GalBSA but apparently delayed GalBSA transfer along successive populations of host organelles. The low density, GalBSA‐containing structures were devoid of proteolytic activity. Anti‐secretory component IgG and F(ab′)2 were selectively excreted into bile, partially or totally as compounds of lower molecular mass. These antibody fragments probably result from a disulfide reduction activity along the pIgA pathway. In conclusion, our data (a) strongly suggest that pIgA and GalBSA are sorted between 3 min and 10 min after injection in non‐lysosomal acidic organelles, (b) identify two successive and physically distinct endosomal populations containing GalBSA, and (c) provide the first evidence for a disulfide reduction activity along the transcytotic pathway of rat hepatocytes.

Variations of endocytic and of lysosomal functions during the cell cycle have been investigated in synchronized hepatoma cells (derived from Morris hepatoma 7288c) by following the cellular uptake of horseradish peroxidase, dextran (mol wt, 70,000), and chloroquine .Cell fractionation and cytochemistry show that in asynchronously growing cells exposed for 1 h to 5 mg/ml peroxidase, the bulk of the enzyme taken up by the cells is found in phagosomes . By using the same experimental system with synchronized HTC cells, large variations of endocytosis are observed during the cell cycle . Peroxidase uptake is lowest during mitosis, increases 5-10 times during G 1 phase, reaches a plateau, and finally decreases at the end of S phase and during G2 phase . A similar evolution is observed for the uptake of dextran (0 .5 or 1 mg/ml), but it is likely that a significant part of the polysaccharide is still associated with the pericellular surface after 1 h . Moreover, dextran is transferred more slowly than peroxidase to lysosomes . Cellular accumulation of chloroquine is related to intralysosomal pH or to the buffering capacity of lysosomes . Our results show that this drug is taken up more rapidly during G 1 and S phases while the rate of accumulation is lowest in mitotic cells .

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.