共查询到20条相似文献,搜索用时 31 毫秒
1.
Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein. 相似文献
2.
Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor 总被引:8,自引:3,他引:5
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
《The Journal of cell biology》1989,109(6):2809-2816
We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells. 相似文献
3.
We have obtained expression of the beta-N-acetylglucosamine-binding receptor from chicken hepatocytes in Xenopus oocytes by injecting mRNA synthesized in vitro from a full length cDNA cloned into an expression vector (Mellow et al: J. Biol Chem 263: 5468-5473, 1988). Immunoprecipitation of the receptor after labeling of oocytes with [35S]-methionine for times ranging from 6 to 72 h revealed 4-5 closely spaced bands of 25-30 kDa after SDS-PAGE. Although these bands were largely resistant to endoglycosidase H cleavage, endoglycosidase F reduced the size of all bands to a single species at 23-24 kDa, indicating that they resulted from heterogeneity in glycosylation of a single polypeptide. Incubation of oocytes expressing this receptor with [125I]-GlcNAc-BSA resulted in 1.8 to 10 x higher levels of cell-associated ligand in mRNA-injected vs. water-injected control oocytes, 2-35% of cell-associated counts was removed by EGTA rinse at 20 degrees C, suggesting that most ligand was inaccessible (presumably intracellular). Immunoprecipitation of sucrose gradient fractions detected receptor molecules predominantly in a light organelle at 1.09-1.12 g/cc (the density of early endosomes and plasma membrane vesicles), with no evidence of the receptor in much heavier yolk platelet fractions even in the presence of ligand. In contrast, internalized [125I]-GlcNAc-BSA was found either at the top of the gradients or in organelles at 1.09-1.17 g/cc and in yolk platelets. TCA precipitation indicated that much intracellular ligand was degraded to acid-soluble fragments. Addition of vitellogenin (the yolk protein precursor) to the medium together with the [125I]-GlcNAc-BSA shifted much of the ligand into yolk platelets. These data indicate that the chicken glycoprotein receptor expressed in oocytes mediates binding and internalization of this ligand into an organelle in which ligand-receptor dissociation occurs, allowing for separation of these two molecules into different compartments. The behavior of ligand in Xenopus oocytes expressing the chicken receptor closely resembles its behavior in hepatocytes. 相似文献
4.
The Kupffer cell receptor for glycoproteins has been reported to have a role in clearance of galactose- and fucose-terminated glycoproteins from circulation. Although the gene and a cDNA encoding the receptor have been described, there has been little study of the receptor protein. To address some questions about possible ligands and functions for this receptor, fragments representing portions of the extracellular domain have been expressed and characterized. The extracellular domain consists of a trimer stabilized by an extended coiled-coil of alpha-helices. The receptor displays monosaccharide-binding characteristics similar to the hepatic asialoglycoprotein receptor, but with somewhat less selectivity. The two best monosaccharide ligands are GalNAc and galactose. alpha-Methyl fucoside is a particularly poor ligand. Analysis of Kupffer cell receptor binding to glycoproteins and oligosaccharides released from them reveals highest affinity for desialylated, complex N-linked glycans. The best glycoprotein ligands contain multiple highly branched oligosaccharides. A human ortholog of the rat receptor gene does not encode a full-length protein and is not expressed in liver. These characteristics suggest that the receptor may have functions parallel to those of the hepatocyte asialoglycoprotein receptor in some (but not all) mammalian species. 相似文献
5.
We have isolated and characterized a cDNA encoding a chicken beta homolog of c-erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene. 相似文献
6.
Meilinger M Gschwentner C Burger I Haumer M Wahrmann M Szollar L Nimpf J Huettinger M 《The Journal of biological chemistry》1999,274(53):38091-38096
Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism. 相似文献
7.
Uptake of mannose-terminated glycoproteins in isolated rat liver cells. Evidence for receptor-mediated endocytosis in hepatocytes.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
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. 相似文献
8.
Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages 总被引:3,自引:0,他引:3
S K Moestrup E I Christensen L Sottrup-Jensen J Gliemann 《Biochimica et biophysica acta》1987,930(3):297-303
125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes. 相似文献
9.
The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C. 相似文献
10.
Differential regulation of glucose transporter isoforms by the src oncogene in chicken embryo fibroblasts. 总被引:4,自引:1,他引:3
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The increase in glucose transport that occurs when chicken embryo fibroblasts (CEFs) are transformed by src is associated with an increase in the amount of type 1 glucose transporter protein, and we have previously shown that this effect is due to a decrease in the degradation rate of this protein. The rate of CEF type 1 glucose transporter biosynthesis and the level of its mRNA are unaffected by src transformation. To study the molecular basis of this phenomenon, we have been isolating chicken glucose transporter cDNAs by hybridization to a rat type 1 glucose transporter probe at low stringency. Surprisingly, these clones corresponded to a message encoding a protein which has most sequence similarity to the human type 3 glucose transporter and which we refer to as CEF-GT3. CEF-GT3 is clearly distinct from the CEF type 1 transporter that we have previously described. Northern (RNA) analysis of CEF RNA with CEF-GT3 cDNA revealed two messages of 1.7 and 3.3 kb which were both greatly induced by src transformation. When the CEF-GT3 cDNA was expressed in rat fibroblasts, a three-to fourfold enhancement of 2-deoxyglucose uptake was observed, indicating that CEF-GT3 is a functional glucose transporter. Northern analyses using a CEF-GT3 and a rat type 1 probe demonstrated that there is no hybridization between different isoforms but that there is cross-species hybridization between the rat type 1 probe and the chicken homolog. Southern blot analyses confirmed that the chicken genomic type 1 and type 3 transporters are encoded by distinct genes. We conclude that CEFs express two types of transporter, type 1 (which we have previously reported to be regulated posttranslationally by src) and a novel type 3 isoform which, unlike type 1, shows mRNA induction upon src transformation. We conclude that src regulates glucose transport in CEFs simultaneously by two different mechanisms. 相似文献
11.
S R Feldman M R Rosenberg K A Ney G Michalopoulos S V Pizzo 《Biochemical and biophysical research communications》1985,128(2):795-802
The binding of 125I-labeled human alpha 2-macroglobulin-methylamine to adult rat hepatocytes in primary culture was studied at 4 degrees C. Cells which had been in culture for 4 hours exhibited steady state ligand binding after 1 hour, a receptor number of 22,400 receptors per cell, and a dissociation constant of 0.6 nM. Adult rat hepatocytes exhibited a significant decrease in receptor number with increased time in primary culture with less than 10% of the initial number of receptors remaining after 2 days (p less than 0.01). In autopsy studies of mice injected intravenously with 125I-labeled alpha 2-macroglobulin-methylamine, greater than 90% of the cleared ligand was found in the liver. Autoradiography of the liver demonstrated that 80% of the ligand was cleared by hepatocytes. It is concluded that the hepatocytes are the primary pathway for clearance from the circulation of receptor recognized alpha 2-macroglobulin. 相似文献
12.
Endocytosis via galactose receptors in vivo : Ligand size directs uptake by hepatocytes and/or liver macrophages 总被引:2,自引:0,他引:2
Jutta Schlepper-Schfer Dorothea Hülsmann Assadollah Djovkar Helmut E. Meyer Lothar Herbertz Hubert Kolb Victoria Kolb-Bachofen 《Experimental cell research》1986,165(2):494-506
We followed the intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues in rat livers. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au5), 17 nm (Au17), 50 nm (Au50) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au5 in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. Using a LacBSA-Au preparation with heterogeneous particle diameter (2.2-11.7 nm) we found that hepatocytes take up only ligands up to the size of 7.8 nm, whereas particles of all sizes available in this experiment are found in liver macrophages and endothelial cells. ASF-Au17 and LacBSA-Au17 are endocytosed by liver macrophages and endothelial cells, but not by hepatocytes. ASF-Au50 is taken up by liver macrophages only. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc. Some 52-65% inhibition was measured in in vivo experiments and 78% inhibition in in situ experiments. GlNAc showed no inhibitory effect. Furthermore, we measured uptake of [125J]ASF and of [125J]ASF adsorbed to Au17 by the different cell populations of rat livers in vivo. While the bulk of the molecular ligand is found in the hepatocyte fraction, the particulate ligand is located in the sinusoidal fraction. 相似文献
13.
Apolipoprotein A-IV. A determinant for binding and uptake of high density lipoproteins by rat hepatocytes 总被引:9,自引:0,他引:9
E Dvorin N L Gorder D M Benson A M Gotto 《The Journal of biological chemistry》1986,261(33):15714-15718
To identify the role of a specific apoprotein other than apoE which might be responsible for the receptor-mediated uptake of high density lipoprotein (HDL) by rat hepatocytes, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) was combined with rat apoE, apoA-I, or apoA-IV to form apoprotein-phospholipid complexes and the complexes were tested for their binding and uptake by primary rat hepatocytes. Apoprotein-POPC complexes were labeled with the specific fluorescent probe, 1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine to monitor their uptake by cultured rat hepatocytes at 37 degrees C using digital fluorescence imaging microscopy or were labeled with 125I to study their binding to hepatocytes at 4 degrees C. POPC, either alone or with apoA-I, was not internalized by rat hepatocytes while complexes containing apoE or apoA-IV were taken up by the cells. Specific binding at 4 degrees C was demonstrated for apoE-free HDL, apoA-IV X POPC, and apoE X POPC but not for apoA-I X POPC. The binding of apoE-free HDL was inhibited by apoA-IV X POPC, apoE-free HDL, and apoA-IV + apoA-I X POPC but not by apoA-I X POPC. Binding of apoA-IV X POPC was inhibited by apoE-free HDL, apoA-IV X POPC, and apoA-IV + apoA-I X POPC, but not by apoE X POPC or apoE-enriched HDL. These data indicate that apoA-IV is a ligand responsible for the rat HDL binding to primary rat hepatocytes and that apoA-IV binds to a receptor site distinct from apoE-dependent receptors such as the apoB,E or chylomicron-remnant receptor. 相似文献
14.
The hepatic binding and uptake kinetics of epidermal growth factor: studies with isolated rat hepatocytes 总被引:1,自引:0,他引:1
Hepatocytes are known to bind and internalize a variety of small molecular weight proteins by a process known as receptor-mediated endocytosis (RME). The purpose of this investigation was to characterize the binding and uptake kinetics of a small protein known to be taken up by the liver by RME, epidermal growth factor (EGF), using suspensions of freshly isolated rat hepatocytes. Rat hepatocytes accumulated 125I-EGF (90 pM) in a temperature-dependent fashion. Isolated hepatocytes incubated at 37 degrees C with 125I-EGF began to release a TCA-soluble radiolabeled material into the incubation medium with a lag period of 20 min. EGF uptake by isolated hepatocytes was linear for only 60 seconds and displayed saturation kinetics (apparent Km of 4 nM and a Vmax of 105 fM/min/10(6) cells). Hepatocytes incubated at 4 degrees C bound, but did not internalize, EGF. Under these conditions, EGF binding was saturable at concentrations above 8 nM. A Scatchard analysis revealed that the average number of receptors per hepatocyte was 7.7 X 10(4) with a dissociation constant of 2.6 nM. These data demonstrate that freshly isolated hepatocytes are capable of binding, internalizing and metabolizing EGF and thus are a good model to study RME of small molecular weight proteins. 相似文献
15.
16.
J E Sewell S K Davis P S Hargis 《Comparative biochemistry and physiology. B, Comparative biochemistry》1989,92(3):509-516
1. Fatty acid binding protein (FABP) was isolated from chicken liver cytosol. 2. Apparent molecular weight, pI, functional activity, and hybridization of a rat hFABP cDNA probe with chicken liver mRNA suggest that chicken liver FABP is structurally related to hepatic FABP (hFABP) previously isolated and characterized in the rat. 3. Fatty acids bound to liver FABP affect the electrophoretic nature of FABP. 4. Levels of liver FABP mRNA isolated from chickens at various stages of development parallel developmental alterations in lipid metabolism, being highest in day old chicks and laying hens versus juvenile birds. 相似文献
17.
The src oncogene can regulate a human glucose transporter expressed in chicken embryo fibroblasts. 总被引:4,自引:1,他引:3
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
When fibroblasts are transformed by the src oncogene, there is a two- to fivefold increase in glucose transport and in the level of immunoprecipitable glucose transporter protein. In chicken embryo fibroblasts (CEFs), this increase is correlated with a comparable reduction in the rate at which the glucose transporter protein is turned over. In contrast, in mammalian fibroblasts glucose transporter biosynthesis is increased by src, but there is little or no change in its turnover. To further understand the action of src on transporter turnover, we investigated whether a mammalian transporter can be stabilized by src in a chicken cell environment. The human type 1 glucose transporter protein (hGT), originally cloned from HepG2 cells, was expressed in CEFs or Rat-1 fibroblasts by using a retroviral vector. In CEFs transformed by a temperature-sensitive src mutant, tsNY68, turnover of hGT was lower at the permissive temperature (36 degrees C) than at the nonpermissive temperature (42 degrees C). When this protein was expressed in CEFs transformed by wild-type src, no difference in turnover was observed at the two temperatures. In the case of Rat-1 cells transformed by the temperature-sensitive src mutant tsLA29, turnover of hGT was the same at the permissive temperature (35 degrees C) as at the nonpermissive temperature (39.5 degrees C). These data demonstrate that a heterologous glucose transporter behaves in the same way in chicken and rat cells as the respective endogenous transporter, i.e., when src is active, the protein is stablilized against turnover in chicken cells but not in rat cells. 相似文献
18.
Identification of a rat liver cDNA and mRNA coding for the 28 kDa gap junction protein 总被引:4,自引:0,他引:4
By screening of a rat liver cDNA library with complex and deoxyinosine containing oligonucleotide probes a cDNA clone was isolated and shown by sequencing to code for the amino-terminal half of the rat liver 28 kDa gap junction protein. The insert hybridized to a 1.9 kb species from rat and mouse liver poly(A)+ RNA in Northern blot analysis. In embryonic mouse hepatocytes the amount of the 1.9 kb mRNA increased 3-fold between 24 and 96 h in culture. This correlates with the previously described increase of the 28 kDa gap junction protein under these conditions. 相似文献
19.
The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37 degrees C) had a 2-fold higher rate of 2-deoxy-D-glucose uptake than the same cells cultured at the non-permissive temperature (41 degrees C). However, both the non-transformed and transformed cells had comparable rates of alpha-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41 degrees C or 37 degrees C, displayed carrier-mediated, intravesicular uptake of D-glucose and alpha-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37 degrees C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41 degrees C. The two types of membrane vesicle had similar uptake rates of alpha-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. Km values for stereospecific D-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37 degrees C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37 degrees C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virally-transformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers. 相似文献
20.
The pathways for fluid phase and receptor mediated endocytosis in rat hepatocytes are different but thermodynamically equivalent 总被引:3,自引:0,他引:3
In isolated rat hepatocytes fluid phase endocytosis, determined by the uptake of the fluorescent dye lucifer yellow (LY), and receptor mediated endocytosis, determined using a ligand for the asialoglycoprotein receptor (asialo-orosomucoid; ASOR), are different pathways based on their different sensitivities to hyperosmolarity induced by sucrose (Oka and Weigel, J. Cell. Biol. 105, 311a, 1987). LY uptake was unaffected by 0.2 M sucrose at all temperatures tested between 12 degrees and 37 degrees C whereas the uptake of 125I-ASOR was completely inhibited at any temperature. Since the two probes are taken up by different pathways it was possible to determine independently the activation energies (Ea) for the fluid phase versus the receptor mediated coated pit endocytic process. The Ea was 26.4 +/- 3.5 and 25.8 +/- 1.9 kcal/mole for, respectively, receptor mediated and fluid phase endocytosis. These values are not significantly different, and we conclude that the fluid phase and receptor mediated pathways are thermodynamically equivalent even though they are independent. 相似文献