Effect on lysosomes of invertase endocytosed by rat-liver

The intracellular localization of invertase endocytosed by rat liver was investigated by analytical centrifugation in sucrose and Percoll gradients of mitochondrial fractions originating from rats killed 15 h after injection. After isopycnic centrifugation in a sucrose gradient, invertase is located in higher density zones than acid hydrolases. The difference between the distribution of invertase and that of acid hydrolases increases with the amount of invertase injected. When the invertase dose is sufficiently high, a change of lysosomal enzyme distribution is clearly visible. It consists in the shift of a proportion of these enzymes to higher density regions where invertase is located. The proportion of hydrolase activity affected by invertase is different for each enzyme measured; it is the least pronounced for acid phosphatase, and most for acid deoxyribonuclease and arylsulfatase. A pretreatment of the rat with Triton WR 1339 considerably decreases the equilibrium density of structures bearing invertase. Nevertheless invertase distribution is quite distinct from that of the bulk of lysosomal enzymes that are recovered in lower density zones of the gradient; on the other hand the invertase injection to rats treated with Triton WR 1339 causes a spreading of the acid hydrolase distribution towards higher density zones. The distribution of acid hydrolases and invertase in a Percoll gradient depends on the sucrose concentration of the solvent. It is shifted towards higher densities when the sucrose concentration increases. The phenomenon is more important for invertase. These results are best explained by supposing that invertase accumulates in a distinct population of lysosomes that can be individualized as a result of the density increase they are subjected to by the invertase they accumulate. It is proposed that these lysosomes mainly originate from non-parenchymal cells of the liver.     

Uptake and intracellular transport in rat liver of formaldehyde-treated bovine serum albumin labelled with 125I-tyramine-cellobiose

1. Endocytosis of formaldehyde-treated bovine serum albumin by rat liver sinusoidal cells has been followed by injecting rats with the protein labelled with 125I-tyramine cellobiose (125I-TCfBSA). 125I-TCfBSA is quickly taken up by the liver; the radioactivity present in the organ reaches a plateau 5-10 min after injection and is maintained for up to at least 180 min. During the first 5 min most of radioactivity remains acid-precipitable. After which, labelled acid-soluble components are produced at a constant rate for up to 30-40 min. 2. Differential centrifugation shows that radioactivity is first recovered mainly in the microsomal fraction. Within a few minutes it exhibits a distribution pattern similar to that of lysosomal enzymes, being chiefly located in the mitochondrial fractions. 3. Isopycnic centrifugation in a sucrose gradient of the microsomal fraction isolated 1 min after injection indicates a similar distribution for radioactivity and alkaline phosphodiesterase. Later, the microsomal radioactivity distribution curve is shifted towards higher densities and becomes distinct from that of the plasma-membrane enzyme. After isopycnic centrifugation in a sucrose gradient of the total mitochondrial fraction a considerable overlapping of acid-precipitable and acid-soluble radioactivity distributions is observed without significant changes with time. The same is observed in a Percoll gradient except that after a relatively long time (greater than 30 min) of injection a marked shift of radioactivity distribution towards higher densities occurs. 4. A pretreatment of rats with Triton WR 1339, a density perturbant of liver lysosomes, causes a striking shift of acid-soluble radioactivity distribution in a sucrose gradient towards lower densities while having markedly less influence on the acid-precipitable distribution. As a result, a distinction between the distribution of both kinds of radioactivity becomes clearly apparent. A preinjection of yeast invertase, modifies the acid-soluble distribution without having a significant effect on the acid-precipitable distribution up to 30 min after 125I-TCfBSA injection. 5. Glycyl-1-phenylalanine-2-naphthylamide largely releases acid-soluble radioactivity associated with the mitochondrial fraction, whatever the time after 125I-TCfBSA injection. On the other hand the proportion of acid-precipitable radioactivity present in the fraction that can be released is almost zero at 10 min after injection, and it later increases. 6. The results presented here are best explained by supposing that, after being trapped in small pinocytic vesicles, 125I-TCfBSA is quickly delivered to the endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

The intracellular distribution of high density lipoproteins taken up by isolated rat hepatocytes

The subcellular distribution of 125I-labelled HDL taken up by rat hepatocytes in vivo and in vitro has been studied with subcellular fractionation techniques: differential centrifugation and isopycnic centrifugation in sucrose gradients. 125I-labelled HDL bind to plasma membranes both in vivo and in vitro and part of the membrane-bound 125I-labelled HDL can be dissociated by the addition of unlabelled HDL. The hepatocytes also internalize 125I-labelled HDL. The 125I-labelled HDL accumulate, however, at different intracellular sites in the in vivo and in vitro situation. The subcellular distribution pattern of 125I-labelled HDL taken up by the cells in vivo is similar to that of the lysosomal marker enzyme acid phosphatase. Peak activity was found at a density of 1.20 g/ml. In vitro 125I-labelled HDL accumulate in an organelle with a medium density of about 1.13 g/ml. This distribution was similar to that of the plasma membrane marker 5'-nucleotidase. The subcellular distribution of radioactivity taken up in vivo was changed to lower density by incubating the cells with chloroquine, a drug known to render the lysosomes more boyant. Chloroquine had no effect on the distribution of 125I-labelled HDL taken up by hepatocytes in vitro.     

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Uptake and intracellular fate of polyethylenimine in vivo

Branched polyamines are extensively used as nonviral vectors for plasmid DNA in transfection experiments. Moreover, recently it has been shown that these compounds are able to eliminate prions from infected cells in cultures. It has been proposed that in both cases endosomes or lysosomes are the site of action. This raises the question of how these molecules are taken up by the cells and what is their intracellular fate. In the work presented here, the question has been addressed by investigating the uptake and the intracellular distribution of branched polyethyleneimine (25 kD) by centrifugation methods. The polyamine was labelled with (125)I-tyramine cellobiose and injected to the rat. The radioactive polymer is taken up after injection into the liver, kidney, spleen, and lungs and remains in these organs for many days. In the liver, it is found mainly in the hepatocytes. Intracellular distribution of radioactivity present in that organ was investigated by differential and isopycnic centrifugations. Early after injection, radioactivity exhibits a distribution pattern similar to that of alkaline phosphodiesterase, a plasma membrane marker. Later, the distribution pattern becomes similar to that of cathepsin C, a lysosomal enzyme. Radioactivity and hydrolase distributions in a sucrose gradient are similarly modified by a pretreatment of the rat with Triton-WR1339, a specific density perturbant of lysosomes. These results indicate that polyethyleneimine is endocytosed and reaches lysosomes. For many days it persists in these organelles probably due to its resistance to lysosomal hydrolases.     

Intracellular transport and degradation of I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment.

The uptake and processing of glucagon into liver endosomes were studied in vivo by subcellular fractionation. After injection of [[125I]iodo-Tyr10]glucagon and [[125I]iodo-Tyr13]glucagon to rats, the uptake of radioactivity into the liver was maximum at 2 min (6% of the dose/g of tissue). On differential centrifugation, the radioactivity in the homogenate was recovered mainly in the nuclear (N), microsomal (P) and supernatant (S) fractions, with maxima at 5, 10 and 40 min, respectively; recovery of radioactivity in the mitochondrial-lysosomal (ML) fraction did not exceed 6% and was maximal at 20 min. On density-gradient centrifugation, the radioactivity associated first (2-10 min) with plasma membranes and then (10-40 min) with Golgi-endosomal (GE) fractions, with 2-5-fold and 20-150-fold enrichments respectively. Subfractionation of the GE fractions showed that, unlike the Golgi marker galactosyltransferase, the radioactivity was density-shifted by diaminobenzidine cytochemistry. Subfractionation of the ML fraction isolated at 40 min showed that more than half of the radioactivity was recovered at lower densities than the lysosomal marker acid phosphatase. Throughout the time of study, the [125I]iodoglucagon associated with the P, PM and GE fractions remained at least 80-90% trichloroacetic acid (TCA)-precipitable, whereas that associated with other fractions, especially the S fraction, became progressively TCA-soluble. On gel filtration and h.p.l.c., the small amount of degraded [125I]iodoglucagon associated with GE fractions was found to consist of monoiodotyrosine. Chloroquine treatment of [125I]iodoglucagon-injected rats caused a moderate but significant increase in the late recovery of radioactivity in the ML, P and GE fractions, but had little effect on the association of the ML radioactivity with acid-phosphatase-containing structures. Chloroquine treatment also led to a paradoxical decrease in the TCA-precipitability of the radioactivity associated with the P and GE fractions. Upon h.p.l.c. analysis of GE extracts of chloroquine-treated rats, at least four degradation products less hydrophobic than intact [125I]iodoglucagon were identified. Radio-sequence analysis of four of these products revealed three cleavages, affecting bonds Ser2-Gln3, Thr5-Phe6 and Phe6-Thr7. When GE fractions containing internalized [125I]iodoglucagon were incubated in iso-osmotic KCl at 30 degrees C, a rapid generation of TCA-soluble products was observed, with a maximum at pH 4. We conclude that endosomes are a major site at which internalized glucagon is degraded, endosomal acidification being required for optimum degradation.     

Fate of injected 125I-labeled cholera toxin taken up by rat liver in vivo. Generation of the active A1 peptide in the endosomal compartment

Subcellular fractionation techniques have been used to assess the localization of injected 125I-labeled cholera toxin (125I-CT) taken up by rat liver in vivo, and to determine whether internalization of the toxin is required for the generation of the active A1 peptide. The uptake of injected 125I-CT into the liver is maximal at 5 min (about 10% injected dose/g). At this time the radioactivity is for the most part recovered in the microsomal (P) fraction, but later on it progressively associates with the mitochondrial-lysosomal (ML) and supernatant fractions. The radioactivity is enriched 7-fold in plasma membranes at 5-15 min, and 15-60-fold in Golgi-endosome (GE) fractions at 15-60 min. On analytical sucrose gradients the radioactivity associated with the P fraction is progressively displaced from the region of 5'-nucleotidase (a plasma membrane marker) to that of galactosyltransferase (a Golgi marker). On Percoll gradients, however, it is displaced towards acid phosphatase (a lysosomal marker). Density-shift experiments, using Triton WR 1339, suggest that some radioactivity associated with the P fraction (at 30 min) and all the radioactivity present in the ML fraction (at 2 h) is intrinsic to acid-phosphatase-containing structures, presumably lysosomes. Comparable experiments using 3,3'-diaminobenzidine cytochemistry indicate that the radioactivity present in GE fractions is separable from galactosyltransferase, and thus is presumably associated with endosomes. The fate of injected 125I-labeled cholera toxin B subunit differs from that of the whole toxin by a more rapid uptake (and/or clearance) of the ligand into subcellular fractions, and a greater accumulation of ligand in the ML fraction. Analysis of GE fractions by SDS/polyacrylamide gel electrophoresis shows that, up to 10 min after injection of 125I-CT, about 80% of the radioactivity is recovered as A subunit and 20% as B subunit, similarly to control toxin. Later on there is a time-dependent decrease in the amount of A subunit and, at least with the intermediate GE fraction, a concomitant appearance of A1 peptide (about 15% of the total at 60 min). In contrast the radioactivity associated with plasma membranes remains indistinguishable from unused toxin. It is concluded that, upon interaction with hepatocytes, 125I-CT (both subunits A and B) sequentially associates with plasma membranes, endosomes and lysosomes, and that endosomes may represent the major subcellular site at which the A1 peptide is generated.     

An Improved Method for the Purification of "Light" Noradrenaline Vesicles from Rat Vas Deferens: Some Biochemical Characteristics

"Light" noradrenaline storage vesicles from nerve endings have been isolated by differential centrifugation and differential gradient centrifugation. They have been further purified by isopycnic sucrose/D2O centrifugation. By using these centrifugation techniques, we obtained an isopycnic gradient fraction in which noradrenaline was enriched about 41 times versus a total homogenate. This factor could be raised to 61 by using seminal ducts of castrated rats. Comparison of the distribution patterns in sucrose/D2O isopycnic gradients indicated that light noradrenaline vesicles of nerve endings contain Mg2+-stimulated ATPase and ATP, but that only a minor part of the dopamine beta-hydroxylase can be associated with these vesicles.     

Intracellular degradation by liver endothelial cells

Investigations were carried out on the intracellular fate of formaldehyde treated bovine serum albumin (F-BSA), in liver non-parenchymal cells. This paper reports the observations and results obtained by us. The first part of our work involved the injecting of the compound into either a) normal rats, b) rats injected with Triton WR 1339 or c) rats treated with mannan. Fractions obtained after differential and isopycnic centrifugation in sucrose gradients, were analysed by SDS-gel electrophoresis and fluorography. The degradation takes place in a two step process. The molecule is first split into radiolabeled compounds that are still acid precipitable. This is followed by the appearance of acid soluble radioactive molecules. In a sucrose gradient the first kind of degradation products exhibit a distribution totally different from that of acid soluble degradation compounds. In the second part of our experiments, fairly pure fractions of the organelles, known to be involved in the endocytic pathway i.e. endosomes, transfer lysosomes and accumulation lysosomes (marked by the presence of either Triton WR 1339 or mannan) were isolated and incubated with [¹²⁵I]-F-BSA. These experiments revealed that endosomes, isolated by us, are incapable of degradation. Accumulation lysosomes arising exclusively from liver non-parenchymal cells (in which mannan had accumulated) though rich in certain hydrolases eg. arylsulfatase did not have an efficient proteolytic machinery. Our results, both fromin vivo andin vitro studies, suggest that the first degradation step occurs in one type of structure (probably not endosomes), a sort of hybrid endosome-lysosome (as they are not affected by glycyl-l-phenyl-2-napthylamide [1]) and the second step in a different type of lysosomes, what we have designated transfer lysosomes.     

The effect of Triton WR-1339 on the subcellular distribution of Trypan Blue and 125I-labelled albumin in rat liver

1. The density-gradient distribution patterns of acid phosphatase, Trypan Blue and denatured (125)I-labelled albumin were studied by discontinuous sucrose- and isopycnic sucrose-density-gradient centrifugation on combined heavy and light mitochondrial (M+L) fractions of liver isolated from normal rats and from rats injected with Triton WR-1339. 2. The results obtained from the subfractionation of the M+L pellet of normal animals indicate that the equilibrium density of Trypan Blue and acid-insoluble radioactivity is the same as that for acid phosphatase, which suggests they are bound by a common membrane to form a distinct subcellular population of lysosomal nature. 3. In contrast, the analysis of the isopycnic gradients obtained on subfractionation of M+L pellets of liver isolated from rats treated with Triton WR-1339 show that the acid-insoluble radioactivity has an equilibrium density around 1.21, whereas the acid hydrolases, including cathepsin D, show the characteristic shift to an equilibrium density of around 1.12. Trypan Blue is distributed along the gradient with distinct peaks at densities 1.22 and 1.12. 4. Similar equilibrium-density distribution patterns were obtained with M+L pellets isolated from rats pretreated with Triton WR-1339 but not injected with Trypan Blue. 5. Treatment of the rats with Triton WR-1339 does not affect albumin digestion of isolated intact lysosomes despite the fact that most of the cathepsin D and the albumin ingested by phagocytosis are located in different vacuoles. 6. It is concluded from these experiments that in the liver of animals treated with Triton WR-1339 (125)I-labelled albumin is located within heterophagosomes which do not fuse with heterolysosomes containing the non-ionic detergent Triton WR-1339. The inability of these two lysosomal populations to fuse is not due to Trypan Blue.     

Contribution of cytoplasmic enzymes to apparent heterogeneity of rat brain mitochondria

Summary The results of this investigation indicate that there is sufficient contamination of washed primary rat brain mitochondrial preparations with cytoplasmic enzymes to cause possible misinterpretation of mitochondrial heterogeneity after isopycnic sucrose density gradient centrifugation, when based solely on enzyme activity measurements. Most of the contamination may be removed by a single 30 min centrifugation using the 3–6 % discontinuous Ficoll gradient method of Clark and Nicklas (1970).     

Influence of starvation,Triton WR-1339 and [131I]-human serum albumin on rat liver lysosomes

The response of rat liver lysosomes to starvation and administration of lysosomotropic agentsviz. Triton WR-1339 and [¹³¹I]-human serum albumin, was assessed in terms of their distribution pattern after isopycnic sucrose density gradient centrifugation. Starvation induced changes in lysosomes appeared to be similar to that produced by the detergent uptake. Both the treatments caused a distinct decline in the equilibration densities of the organelles. On the other hand, injected labelled protein failed to comigrate with the lysosomal markers in starved as well as Triton treated rats and conspicuously remained in a region of high specific gravity in the gradient. These findings indicate retarded fusion between secondary lysosomes and [¹³¹I]-human serum albumin containing phagosomes in the livers of rats subjected to starvation or detergent treatment     

Studies on ethylene binding by cell-free preparations from cotyledons of Phaseolus vulgaris L.: subcellular localization

Abstract. Studies on the subcellular location of ethylene binding activity from developing cotyledons of Phaseolus vulgaris L. are described. Binding activity has been shown to be predominantly membrane bound. When separated by rate-zonal centrifugation more than 70% of this activity was of low sedimentation rate. The slowly sedimenting band of activity was further fractionated into three bands by isopycnic density gradient centrifugation. The three bands occur at sucrose densities of 1.125 g cm⁻³, 1.155 g cm⁻³ and 1.175 g cm⁻³, corresponding to the distribution of putative marker enzymes for the cell endomembrane system and to protein body membranes. Further circumstantial evidence was obtained by electron microscopy and sucrose step gradient centrifugation.     

Analysis by isopycnic centrifugation of isolated nucleoids of Escherichia coli.

The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 +/- 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 +/- 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.     

Uptake and subcellular distribution of injected transferrin in rat liver

Radioactively labelled transferrin was injected into rats intravenously and its uptake and subcellular distribution in the liver was investigated. The amount of radioactivity in the liver remained constant from 10 min after injection. It was not influenced by asialoglycoproteins. The radioactive label was identified as asialotransferrin. After subcellular fractionation by differential and zonal sucrose density-gradient centrifugation the label was enriched in a low-density endocytic compartment showing fluorescence quenching of acridine orange and N-ethylmaleimide-sensitive ATPase activity. The data fit into a model of continuous transferrin-receptor-mediated recycling through the hepatocyte's endocytic compartment.     

Differences in the cellular location of substances endocytosed by rat liver as observed from the distribution patterns obtained after isopycnic centrifugation in a sucrose gradient

We have investigated the distribution of several substances endocytosed by rat-liver, after isopycnic centrifugation in a sucrose gradient of the MLP fractions (de Duve, Pressman, Gianetto, Wattiaux and Appelmans (1955) Biochem.J. 63, 604-617) isolated at increasing times after injection. It has been observed that there are changes in the distribution pattern with time depending on whether the substance is taken up by parenchymal or sinusoidal cells. The results suggest that centrifugation experiments can be informative with respect to the cellular location of a molecule endocytosed by the liver.     

Uptake of mannose-terminated glycoproteins in isolated rat liver cells. Evidence for receptor-mediated endocytosis in hepatocytes.

Even though most of the hepatic binding capacity for mannose-terminated glycoproteins has previously been shown to reside in the hepatocytes (not in the non-parenchymal cells), detailed evidence for the specific uptake of mannose-terminated ligands has been lacking. In the present studies, yeast invertase, a large glycoprotein (Mr 270 000) containing about 50% mannose, was shown to be taken up into hepatocytes by receptor-mediated endocytosis. The uptake was saturable and could be specifically inhibited by mannosides or by a Ca2+ chelator. The asialo-glycoprotein receptor was not involved. The low-Mr (13 000) ligand ribonuclease B, which contains a single high-mannose glycan, was not taken up by hepatocytes; however, it was taken up as fast as invertase by non-parenchymal liver cells. After injection of 131I-invertase into a rat in vivo, about one-half of the labelled protein was recovered in the hepatocytes. On a per-cell basis, each endothelial cell contained 3-4 times as much radioactivity as did the hepatocytes. On fractionation of hepatocytes in sucrose gradients, invertase showed a different intracellular distribution from that of asialo-fetuin, in that invertase moved much faster into that region of the gradient where the lysosomes were recovered. This indicates that invertase and asialo-fetuin are not transported intracellularly by identical mechanisms.     

Purification of eukaryotic ribosomes by isopycnic centrifugation in sucrose

A simple and inexpensive procedure for the isolation and purification of ribosomes from eukaryotes is described. The method avoids pelleting of ribosomes at high centrifugal forces and involves isopyenic centrifugation of the post-mitochrondrial supernatant in sucrose, precipitation of ribosomes with 10% polyethylene glycol, and zonal sucrose gradient centrifugation. The ribosomes obtained in this way are very pure and thus especially suited for the measurements of physical properties. The isopycnic centrifugation can also be used for the purification of other macromolecules and is only limited by a maximum density of sucrose of 1.40 g/cm³ obtained at the bottom of the centrifugation tubes.     

Nachweis und Lokalisation von Tryptophan-Synthetase in Samen von Juglans regia L.

Summary Cell-free extracts from seeds of Juglans regia synthesize tryptophan from L-serine and indole. Tryptophan synthetase has maximal activity in the range between pH 7 and 8. The enzyme is associated with a particulate fraction (density 1,210 g/ml) which is separated from the mitochondria (density 1,191 g/ml) after isopycnic density centrifugation on a continuous sucrose gradient.