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1.
A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished. 相似文献
2.
H Akiyama K Shii K Yokono K Yonezawa S Sato K Watanabe S Baba 《Biochemical and biophysical research communications》1988,155(2):914-922
Although insulin-degrading enzyme (IDE) has been implicated in the intracellular degradation of insulin, the cellular localization of this enzyme is still controversial. In the present study, we have examined the cellular localization of IDE in the rat liver by three different techniques using monoclonal antibodies. First, direct immunohistochemical staining of rat liver with one of the monoclonal antibodies revealed that IDE immunoreactivity mainly exists in parenchymal cells, especially in the vicinity of the portal tract and also in the epithelium of the bile duct under light microscopy. In the electron microscopic study, IDE immunoreactivity was found in the cytoplasm near the rough endoplasmic reticulum but not in the plasma membrane, nucleus, or mitochondria. Second, immunoblotting analysis of the subcellular fraction in rat liver showed that the monoclonal antibody specifically reacted with a single polypeptide in the cytosolic fraction, of apparent Mr 110,000, which was consistent with the Mr of IDE. However, a polypeptide band corresponding to IDE could not be observed in the plasma membrane, mitochondrial, or lysosomal fraction. Third, IDE was only detectable in the cytosolic fraction by sandwich radioimmunoassay using two monoclonal antibodies. These results all suggest that IDE is a cytosolic enzyme. 相似文献
3.
The intracellular distribution of lysosomal enzymes in lymphocytes has previously been only poorly defined, mainly by cytochemical procedures of low resolution. In the present study we have used a post-embedding immunogold technique to identify the precise ultrastructural localization of a lysosomal enzyme, beta-glucuronidase, in activated lymphocytes embedded in Lowicryl K4M resin. We show that this enzyme is present in the rough endoplasmic reticulum, in the Golgi complex, and in vesicular organelles which probably include lysosomes. 相似文献
4.
Our recent studies have shown that cathepsin L is first synthesized as an enzymatically inactive proform in endoplasmic reticulum and is successively converted into an active form during intracellular transport and we postulated that aspartic proteinases would be responsible for the intracellular propeptide-processing step of procathepsin L accompanied by the activation of enzyme (Y. Nishimura, T. Kawabata, and K. Kato (1988) Arch. Biochem. Biophys. 261, 64-71). To better understand this proposed mechanism, we investigated the effect of pepstatin, a potent inhibitor of aspartic proteinases, on the intracellular processing kinetics of cathepsin L analyzed by pulse-chase experiments in vivo with [35S]methionine in the primary cultures of rat hepatocytes. In the pepstatin-treated cells, the proteolytic conversion of cellular procathepsin L of 39 kDa to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 5-h chase periods. Further, the subcellular fractionation experiments demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the light density microsomal fraction after 2 h of chase. These results suggest that pepstatin treatment caused a significant inhibitory effect on the intracellular processing and also on the intracellular movement of procathepsin L from the endoplasmic reticulum to the lysosomes. These findings provide the first evidence showing that aspartic proteinase may play an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo. Therefore, we suggest that cathepsin D, a major lysosomal aspartic proteinase, is more likely to be involved in this proposed model in the lysosomes. 相似文献
5.
Intracellular transport and processing of lysosomal cathepsin B 总被引:2,自引:0,他引:2
Intracellular transport and processing of lysosomal cathepsin B was investigated in the subcellular fractions of rat liver by pulse-labeling experiments with [35S]methionine in vivo. A newly synthesized procathepsin B with a molecular weight of 39 kDa firstly appeared in the rough microsomal fraction at 10 min postinjection of label. This procathepsin B moved from the microsomal fractions to the Golgi subfractions at 30 min postinjection, and then a processed mature enzyme appeared in the lysosomal fraction at 60 min. These results suggest that the propeptide-processing of procathepsin B takes place in lysosomes in the course of intracellular transport from endoplasmic reticulum through Golgi complex to lysosomes. 相似文献
6.
Epoxide hydrolase is a marker for the smooth endoplasmic reticulum in rat liver. 总被引:6,自引:0,他引:6
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Epoxide hydrolase (EH, EC 3.3.2.3) was chosen as a potential marker for smooth endoplasmic reticulum, because this enzyme is inducible by drugs such as phenobarbital. The hypothesis was verified in rat liver using immunochemical and immunocytochemical techniques. Antibodies were raised to the purified protein. These antibodies were affinity purified using the enzyme immobilized on Sepharose Ultrogel. The specificity of the antibodies was assayed by immunoelectrotransfer (Western blot). The labelling of rat liver thin frozen sections with protein A-gold particles demonstrated that the antibodies specifically recognised smooth endoplasmic reticulum membranes. Rough endoplasmic reticulum, other intracellular organelles and plasma membrane were unlabelled. 相似文献
7.
Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver 总被引:24,自引:0,他引:24
An alpha-mannosidase activity has been identified in a preparation of rat liver endoplasmic reticulum and shown to be distinct from the previously described Golgi alpha-mannosidases I and II and the lysosomal alpha-mannosidase. The enzyme was solubilized with deoxycholate and separated from other alpha-mannosidases by passage over concanavalin A-Sepharose to which it does not bind. The endoplasmic reticulum alpha-mannosidase cleaves alpha-1,2-linked mannoses from high mannose oligosaccharides and, unlike Golgi alpha-mannosidase I, is active against p-nitrophenyl-alpha-D-mannoside (Km = 0.17 mM). It has no activity toward GlcNAc-Man5GlcNAc2 peptide, the specific substrate of the Golgi alpha-mannosidase II. The endoplasmic reticulum alpha-mannosidase activity toward p-nitrophenyl-alpha-D-mannoside is relatively insensitive to swainsonine, an inhibitor of both the lysosomal alpha-mannosidase and Golgi alpha-mannosidase II. We propose that the endoplasmic reticulum alpha-mannosidase is responsible for the removal of mannose residues from asparagine-linked high mannose type oligosaccharides prior to their entry into the Golgi. 相似文献
8.
Kozlov AV Gille L Miller I Piskernik C Haindl S Staniek K Nohl H Bahrami S Ohlinger W Gemeiner M Redl H 《Biochemical and biophysical research communications》2007,352(1):91-96
In this study, we determined functional integrity and reactive oxygen species generation in mitochondria and endoplasmic reticulum in liver of rats subjected to endotoxic shock to clarify whether intracellular reactive oxygen species (ROS) destabilize cellular integrity causing necrosis in rats challenged with lipopolysaccharide (LPS). LPS caused drastically increased plasma levels of alanine aminotransferase, suggesting damage to plasma membranes of liver cells. Liver necrosis was confirmed by histological examination. LPS induced a significant increase in ROS production in rat liver mitochondria (RLM), but did not impair mitochondrial function. In contrast to mitochondria, enzymatic activity and ROS production of cytochrome P450 were lower in microsomal fraction obtained from LPS-treated animals, suggesting the dysfunction of endoplasmic reticulum. Protein patterns obtained from RLM by two-dimensional electrophoresis showed significant upregulation of mitochondrial superoxide dismutase by LPS. We hypothesize that upregulation of this enzyme protects mitochondria against mitochondrial ROS, but does not protect other cellular compartments such as endoplasmic reticulum and plasma membrane causing necrosis. 相似文献
9.
Immunocytochemical localization of lysosomal acid phosphatase in normal and "I-cell" fibroblasts 总被引:1,自引:0,他引:1
G Parenti R Willemsen A T Hoogeveen M Verleun-Mooyman J M Van Dongen H Galjaard 《European journal of cell biology》1987,43(1):121-127
This study represents the first example of immunological localization of lysosomal acid phosphatase. The intracellular localization of lysosomal acid phosphatase was investigated with immunocytochemical methods at the light and electron microscopical level in cultured fibroblasts obtained from normal subjects and from a patient with I-cell disease. Double-labeling studies using fluorescence microscopy showed that acid phosphatase is present in the same organelles as other hydrolases. At the electron microscopic level in control fibroblasts acid phosphatase was found in the rough endoplasmic reticulum, lysosomes, at the plasma membrane, in vesicles just below the plasma membrane and in multivesicular bodies. This localization was comparable with that of other lysosomal enzymes tested (acid alpha-glucosidase, N-acetyl-beta-hexosaminidase, beta-galactosidase). Acid phosphatase labeling was mainly found in association with the lysosomal membrane and with membranous material present within the lysosome. In I-cell fibroblasts the label was present in the same subcellular organelles but always associated with membranous structures. We suggest that the association of acid phosphatase with membranes might explain the normal enzyme activity found in I-cell fibroblasts. 相似文献
10.
Summary The intracellular distribution of lysosomal enzymes in lymphocytes has previously been only poorly defined, mainly by cytochemical procedures of low resolution. In the present study we have used a post-embedding immunogold technique to identify the precise ultrastructural localization of a lysosomal enzyme, -glucuronidase, in activated lymphocytes embedded in Lowicryl K4M resin. We show that this enzyme is present in the rough endoplasmic reticulum, in the Golgi complex, and in vesicular organelles which probably include lysosomes. 相似文献
11.
F Umezawa Y Eto T Tokoro F Ito K Maekawa 《Biochemical and biophysical research communications》1985,127(2):663-667
Enzyme replacement with liposomes containing beta-galactosidase obtained from charonia lumpas was carried out in murine globoid cell leukodystrophy (GLD). Charonia lumpas beta-galactosidase was able to hydrolyze galactocerebroside trapped into liposomes prepared from lecithin, cholesterol and sulfatide (molar ratio; 7:2:1). Liposomes containing charonia lumpas beta-galactosidase were successfully incorporated into the mouse tissues. 3H-galactocerebroside labeled liposomes were also incorporated into mouse liver, spleen and other tissues. The accumulation rate of 3H-galactocerebroside into twithcer mice liver and spleen was almost 40 to 100 times higher than those of controls and degraded to 70 to 80% of accumulated radioactivity of 3H-galactocerebroside by single injection of liposomes containing charonia lumpas beta-galactosidase. Results suggest that exogeneous enzyme trapped in liposomes can be useful for the correction of accumulated compound. 相似文献
12.
Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes 总被引:34,自引:0,他引:34
H D Fischer A Gonzalez-Noriega W S Sly D J Morré 《The Journal of biological chemistry》1980,255(20):9608-9615
beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes. 相似文献
13.
An Arthrobacter beta-galactosidase has homology with the lysosomal acid beta-galactosidases from humans and mice and with a Xanthomonas manihotis enzyme. Phylogenetic analysis of the deduced amino acid sequence showed an unusual pattern, with this procaryotic enzyme clustering within the animal clade. The gene encodes a subunit of 52 kDa, and the enzyme appears to be active as a dimer. The enzyme hydrolyzed substrates with either a beta-1,4 or a beta-1,3 linkage. 相似文献
14.
The generation of enzymes located in lysosomes, in cytosol or in endoplasmatic reticulum/Golgi complex is studied in heterokaryons in which chick erythrocyte nuclei are reactivated. The lysosomal enzymes, alpha-glucosidase (alpha-glu) and beta-galactosidase (beta-gal), are synthesized in heterokaryons obtained after fusion of chick erythrocytes with human fibroblasts of patients with Pompe's disease (alpha-glu-deficient) and GM1-gangliosidosis (beta-gal-deficient), respectively. The enzymes appear to be of chick origin and their activities can be detected at first around 4 days after fusion, i.e., at a time when the nucleoli in the erythrocyte nuclei have been reactivated. Maximal activities are reached around 15 days after fusion. No generation of the lysosomal enzyme beta-hexosaminidase is detected in the heterokaryons up to 23 days after fusion of chick erythrocyte with either beta-hexosaminidase A- and B-deficient fibroblasts (Sandhoff's disease) or beta-hexosaminidase A-deficient fibroblasts (Tay-Sachs disease). Similarly no expression of the cytosol enzyme glucose-6-phosphate dehydrogenase (G6PD) is fond up to 30 days after fusion, when chick erythrocytes are fused with fibroblasts from two different G6PD-deficient cell strains (residual activities of 4 and 20% respectively). Indirectly we examined N-acetyl-glucosamine-1-phosphate transferase activity, an enzyme located in the endoplasmic reticulum/Golgi region. This enzyme is needed for the phosphorylation of the lysosomal hydrolases and absence of its activity is the cause of the multiple lysosomal enzyme deficiencies in patients with I-cell disease. The retention of both, chick and human beta-galactosidase in the experiments in which I-cell fibroblasts were fused with chick erythrocytes indicates a reactivation of the gene coding for this phosphorylating enzyme. It also implies that this step in the processing of human lysosomal enzymes is not species-specific. 相似文献
15.
Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver 总被引:11,自引:0,他引:11
M P Reinhart J T Billheimer J R Faust J L Gaylor 《The Journal of biological chemistry》1987,262(20):9649-9655
We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER. 相似文献
16.
D Fekkes E van Overmeeren-Kaptein R Docter G Hennemann T J Visser 《Biochimica et biophysica acta》1979,587(1):12-19
The iodothyronine-deiodinating enzymes (iodothyronine-5- and 5'-deiodinase) of rat liver were found to be located in the parenchymal cells. Differential centrifugation of rat liver homogenate revealed that the deiodinases resided mainly in the microsomal fraction. The subcellular distribution pattern of these enzymes correlated best with glucose-6-phosphatase, a marker enzyme of the endoplasmic reticulum. Plasma membranes, prepared by discontinuous sucrose gradient centrifugation, were found to contain very little deiodinating activity. Analysis of fractions obtained during the course of plasma membrane isolation showed that the deiodinases correlated positively with glucose-6-phosphatase (r larger than or equal to 0.98) and negatively with the plasma membrane marker 5'-nucleotidase (r ranging between -0.88 and -0.97). It is concluded that the iodothyronine-deiodinating enzymes of rat liver are associated with the endoplasmic reticulum. 相似文献
17.
Hans Glaumann Bengt Arborgh Torsten Lindeborg 《Virchows Archiv. B, Cell pathology including molecular pathology》1977,23(1):17-28
Phenobarbital was given to male rats as a single injection and as repetitive injections for 7 days. The effects of treatment on the lysosomal hydrolases acid phosphatase, cathepsin D, and aryl sulfatase were analyzed at different intervals ranging from 1 to 15 days after seven injections, and from 1 to 48 h after a single injection. In both cases, microsomal protein and NADPH-cytochrome c reductase were measured to ensure proper induction. After a single injection, a slight decrease in hydrolytic activities was observed. Repetitive administration of phenobarbital gave rise to a marked decrease of lysosomal enzyme activities 1 day after cessation of treatment. This decrease was followed by a continuous increase in activity up to day 3 and 4. One or 2 weeks after treatment, enzyme activities declined to control values. The increase in activity of lysosomal hydrolytic enzymes was correlated with the onset of induced autophagy of endoplasmic reticulum membranes described as occurring in liver upon cessation of phenobarbital exposure. It is concluded that phenobarbital treatment per se decreases lysosomal enzyme activities, whereas the induced autophagy following cessation of exposure is associated with enhanced levels of lysosomal hydrolases in rat liver. 相似文献
18.
Sialylation is a biosynthetic process occurring in the trans compartments
of the Golgi apparatus. Corresponding evidence is based on localization and
biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as
previously reported. Here we describe generation and characterization of
polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase
(ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a
beta-galactosidase-human-ST3Gal III fusion- protein from E.coli ,
respectively. These antibodies were used to localize ST3Gal III by
immunofluorescence in various cell lines and rat kidney tissue sections. In
transiently transfected COS cells the antibodies directed to soluble
sialyltransferase or the sialyltransferase portion of the fusion-protein
only recognized the recombinant antigen retained in the endoplasmic
reticulum. However, an antibody fraction crossreactive with
beta-galactosidase recognized natively expressed ST3Gal III which was found
to be colocalized with beta1, 4-galactosyltransferase in the Golgi
apparatus of several cultured cell lines. Antibodies affinity purified on
the beta- galactosidase-ST3Gal III fusion-protein column derived from both
antisera have then been used to localize the enzyme in perfusion-fixed rat
kidney sections. We found strong staining of the Golgi apparatus of tubular
epithelia and a brush-border-associated staining which colocalized with
cytochemical staining of the H+ATPase. This subcellular localization was
not observed for ST6Gal I which localized to the Golgi apparatus. These
data show colocalization in the Golgi apparatus and different post-Golgi
distributions of the two sialyltransferases.
相似文献
19.
The UDP-N-acetylglucosamine-asparagine-sequon N-acetyl-beta-D-glucosaminyltransferase activity in preparations of rough endoplasmic reticulum from regenerating rat liver. 总被引:3,自引:2,他引:1
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The UDP-N-acetylglucosamine-asparagine-sequon N-acetylglucosaminyltransferase activity in preparations of rough endoplasmic reticulum from rat liver is less than 1% of that in preparations from rabbit liver. The activity of the enzyme is increased about 20-fold in preparations from regenerating rat liver within 48h after partial hepatectomy. A smaller, but still marked, increase (8-10-fold) occurs in preparations of rough endoplasmic reticulum from sham-operated rats. 相似文献
20.
Choline deficiency causes translocation of CTP:phosphocholine cytidylyltransferase from cytosol to endoplasmic reticulum in rat liver 总被引:2,自引:0,他引:2
The choline-deficient rat liver has been chosen as a physiologically relevant model system in which to study the regulation of phosphatidylcholine biosynthesis. When 50-g rats were placed on a choline-deficient diet for 3 days, the activity of CTP:phosphocholine cytidylyltransferase (CT) was increased 2-fold in the microsomes and decreased proportionately in the cytosol. A low titer antibody to CT was obtained from chickens and used to identify the amount of CT protein in cytosol from rat liver. The amount of CT recovered from the choline-deficient cytosol was significantly less than in cytosol from choline-supplemented rats. When hepatocytes were prepared from choline-deficient livers, supplementation of the medium of the cells with choline caused CT to move from the membranes to cytosol within 1-2 h. The activity of another translocatable enzyme of glycerolipid metabolism, phosphatidate phosphohydrolase, was unchanged in cytosol from choline-deficient rat livers, and the microsomal activity of this enzyme was only minimally increased. When the livers were fractionated into endoplasmic reticulum and Golgi, there was a 2-fold increase in the activity on the endoplasmic reticulum from choline-deficient livers but no change in activity associated with Golgi. Thus, the increased association of CT with endoplasmic reticulum in choline-deficient livers appears to be specific to that subcellular fraction, and the subcellular location of other enzymes may not be affected. 相似文献