Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 39-kDa protein copurifies with the low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor. We recently utilized glutathione S-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (Kd approximately 8-10 nM). These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue-type plasminogen activator (t-PA) and alpha 2-macroglobulin-methylamine (alpha 2M*) binding whereas GST/115-319 only potently inhibits t-PA binding. Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding. In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and alpha 2M* binding to LRP. The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined. Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and alpha 2M* binding. These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is required for inhibition of t-PA and alpha 2M* binding as well as for the direct binding of amino-terminal constructs to LRP. Within carboxy-terminal domains 200-225 and 311-319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding. Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of alpha 2M* binding. Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.
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PMID:Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein. 753 37

Periportal and perivenous rat liver parenchymal cells were isolated according to the digitonin-collagenase perfusion method. Affinities and maximal specific binding of a conjugate of glutathione S-transferase and the alpha 2-macroglobulin receptor-associated protein (GST-39kDaP), of lactoferrin and of transferrin to freshly isolated periportal parenchymal cells in vitro were not significantly different from values obtained with perivenous cells. It is concluded that the receptors for these three ligands show a zonally homogeneous expression in rat liver. The zonal homogeneity in binding observed for GST-39kDaP is at variance with the 1.5-fold higher periportal over perivenous binding of trypsin-activated alpha 2-macroglobulin. Since GST-39kDaP as well as trypsin-activated alpha 2-macroglobulin are ligands for the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein, it is suggested that GST-39kDaP can bind to (an) additional receptor(s) with a higher perivenous expression. The zonal homogeneity observed with lactoferrin, an inhibitor of ligand binding to the lipoprotein remnant receptor, may indicate zonal homogeneity of the lipoprotein remnant receptor. The observed zonal homogeneity of the transferrin receptor suggests an equal and essential need for iron by parenchymal cells across the rat liver acinus in vivo.
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PMID:Zonal distribution of receptor binding of trypsin-activated alpha 2-macroglobulin, alpha 2-macroglobulin receptor-associated protein, lactoferrin and transferrin on rat liver parenchymal cells. 754 60

The ligand-binding domain of the low-density lipoprotein receptor comprises seven cysteine-rich repeats, which have been highly conserved through evolution. This domain mediates interactions of the receptor with two lipoprotein apoproteins, apo E and apo B-100, putatively through a calcium-dependent association of the ligands with a cluster of acidic residues on the receptor. The second repeat (rLB2) of the receptor binding domain has been expressed as a thrombin-cleavable GST fusion protein, cleaved, and purified. On oxidation the protein refolded to give a single peak on reverse-phase HPLC. The aqueous solution structure of rLB2 has been determined using two-dimensional 1H NMR spectroscopy. In contrast to the amino-terminal repeat, rLB1, rLB2 has a very flexible structure in water. However, the conformation of rLB2 is markedly more ordered in the presence of a 4-fold molar excess of calcium chloride; the proton resonance dispersion and the number of NOESY cross-peaks are greatly enhanced. The three-dimensional structure of rLB2, obtained from the NMR data by molecular geometry and restrained molecular dynamics methods, parallels that of rLB1, with an amino-terminal hairpin structure followed by a succession of turns. However, there are clear differences in the backbone topology and structural flexibility. As for rLB1, the acidic residues are clustered on one face of the module. The side chain of Asp 37, which is part of a completely conserved SDE sequence thought to be involved in ligand binding, is buried, as is its counterpart (Asp 36) in rLB1. These results provide the first experimental support for the hypothesis that each of the repeats in the ligand-binding domain has a similar global fold but also highlight significant differences in structure and internal dynamics.
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PMID:Three-dimensional structure of the second cysteine-rich repeat from the human low-density lipoprotein receptor. 757 52

The catabolism of the novel plasminogen activator reteplase (BM 06.022) was described. For this purpose BM 06.022 was radiolabelled with 125I or with the accumulating label 125I-tyramine cellobiose (125I-TC). BM 06.022 was injected at a pharmacological dose of 380 micrograms/kg b.w. and it was cleared from the plasma in a biphasic manner with a half-life of about 1 min in the alpha-phase and t1/2 of 20-28 min in the beta-phase. 28% and 72% of the injected dose was cleared in the alpha-phase and beta-phase, respectively. Initially liver, kidneys, skin, bones, lungs, spleen, and muscles contributed mainly to the plasma clearance. Only liver and the kidneys, however, were responsible for the uptake and subsequent degradation of BM 06.022 and contributed for 75% to the catabolism of BM 06.022. BM 06.022 was degraded in the lysosomal compartment of both organs. Parenchymal liver cells were responsible for 70% of the liver uptake of BM 06.022. BM 06.022 associated rapidly to isolated rat parenchymal liver cells and was subsequently degraded in the lysosomal compartment of these cells. BM 06.022 bound with low-affinity to the parenchymal liver cells (550 nM) and the binding of BM 06.022 could be displaced by t-PA (IC50 5.6 nM), indicating that the low-density lipoprotein receptor-related protein (LRP) could be involved in the binding of BM 06.22. GST-RAP, which is an inhibitor of LRP, could in vivo significantly inhibit the uptake of BM 06.022 in the liver. It is concluded that BM 06.22 is metabolized primarily in the liver and the kidneys. These organs take up and degrade BM 06.022 in the lysosomes. The uptake mechanism of BM 06.022 in the kidneys is unknown, while LRP is responsible for a low affinity binding and uptake of BM 06.022 in parenchymal liver cells.
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PMID:Uptake, internalization and degradation of the novel plasminogen activator reteplase (BM 06.022) in the rat. 877 28

The nature of the liver binding site which is responsible for the initial recognition and clearance of chylomicron-remnants and beta-migrating very-low-density lipoprotein (beta-VLDL) is under active dispute. We have investigated the effect of the 39-kDa receptor-associated protein (RAP) on the recognition site for activated alpha 2-macroglobulin and beta-VLDL on rat liver parenchymal cells in vivo and in vitro in order to analyze whether both substrates are recognized and internalized by the same receptor system. Radiolabelled trypsin-activated alpha 2-macroglobulin (alpha 2M-T) was cleared rapidly by the liver (maximal uptake of 80.8 +/- 1.0% of the injected dose). Prior injection of 5, 15, or 50 mg gluthathione-S-transferase-linked RAP (GST-RAP)/kg rat reduced the liver uptake to 62.2 +/- 2.3%, 59.3 +/- 1.1%, or 2.9 +/- 0.1% of the injected dose, respectively. Concurrently the serum decay was strongly delayed after injection of 50 mg GST-RAP/kg rat but this did not affect the serum decay and liver uptake of 125I-beta-VLDL. Binding studies with isolated liver parenchymal cells in vitro demonstrated that the binding of 125I-alpha 2M-T was 98% inhibited by GST-RAP with an IC50 of 0.3 microgram/ml (4.2 nM), whereas the binding of 125I-beta-VLDL and 125I-beta-VLDL + recombinant apolipoprotein E (rec-apoE) was unaffected by GST-RAP up to 50 micrograms/ml (700 nM). Also, the cell association and degradation of alpha 2M-T was blocked by RAP, while the association and degradation of beta-VLDL and beta-VLDL + rec-apoE were not influenced. The inhibitory effect of RAP on the cell association and degradation of alpha 2M-T lasted for 1-2 h of incubation at 37 degrees C. The binding of the radioiodinated RAP to isolated liver parenchymal cells was highly efficiently coupled to lysosomal degradation. Upon in vivo injection into rats, 125I-labeled RAP is rapidly cleared from the serum and taken up by the liver, which is also coupled to efficient degradation. Since RAP blocks binding of all known ligands to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (the alpha 2Mr/LRP) and at high concentrations the binding to the LDL receptor, we conclude that the initial binding and internalization of beta-VLDL by rat liver parenchymal cells is not mediated by the alpha 2Mr/LRP. The properties of binding of beta-VLDL to rat liver parenchymal cells points to an apoE-specific recognition site for lipoprotein remnants which differs from the alpha 2Mr/LRP, proteoglycans and the LDL receptor and is tentatively called the lipoprotein remnant receptor.
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PMID:Blockade of the alpha 2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein on rat liver parenchymal cells by the 39-kDa receptor-associated protein leaves the interaction of beta-migrating very-low-density lipoprotein with the lipoprotein remnant receptor unaffected. 902

Megalin (gp330) is a member of the low-density lipoprotein receptor gene family. Like other members of the family, it is an endocytic receptor that binds a number of specific ligands. Megalin also binds the receptor-associated protein (RAP) that serves as an exocytic traffic chaperone and inhibits ligand binding to the receptor. To investigate the fate of megalin/RAP complexes, we bound RAP glutathione-S-transferase fusion protein (RAP-GST) to megalin at the surface of L2 yolk sac carcinoma cells and followed the trafficking of the complexes by immunofluorescence and immunogold labeling and by their distribution on Percoll gradients. We show that megalin/RAP-GST complexes, which are internalized via clathrin-coated pits, are delivered to early endosomes where they accumulate during an 18 degrees C temperature block and colocalize with transferrin and transferrin receptor. Upon release from the temperature block, the complexes travel to late endosomes where they colocalize with rab7 and can be coprecipitated with anti-RAP-GST antibodies. Dissociation of the complex occurs in late endosomes and is most likely triggered by the low pH (approximately 5.5) of this compartment. RAP is then rapidly delivered to lysosomes and degraded whereas megalin is recycled to the cell surface. When the ligand, lipoprotein lipase, was bound to megalin, the receptor was found to recycle through early endosomes. We conclude that in contrast to receptor/ligand complexes, megalin/RAP complexes traffic through late endosomes, which is a novelty for members of the low-density lipoprotein receptor gene family.
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PMID:Endocytic trafficking of megalin/RAP complexes: dissociation of the complexes in late endosomes. 918 2

Several clinical studies have demonstrated an inverse relationship between circulating levels of estrogen and tissue-type plasminogen activator (t-PA). The present study was designed to test the hypothesis that estrogens lower plasma levels of t-PA by increasing its clearance from the bloodstream. 17alpha-Ethinyl estradiol (EE) treatment resulted in a significant increase in the clearance rate of recombinant human t-PA in mice (0.46 mL/min in treated mice v 0. 32 mL/min in controls; P <.01). The clearance of endogenous, bradykinin-released t-PA in rats was also significantly increased after EE treatment (area under the curve [AUC], 24.9 ng/mL. min in treated animals v 31.9 ng/mL. min in controls; P <.05). Two distinct t-PA clearance systems exist in vivo: the low-density lipoprotein receptor-related protein (LRP) on liver parenchymal cells and the mannose receptor on mainly liver endothelial cells. Inhibition of LRP by intravenous injection of receptor-associated protein (RAP) as a recombinant fusion protein with Salmonella japonicum glutathione S-transferase (GST) significantly retarded t-PA clearance in control mice (from 0.41 to 0.25 mL/min; n = 5, P <.001) and EE-treated mice (from 0.66 to 0.35 mL/min; n = 5, P <.005), but did not eliminate the difference in clearance capacity between the 2 experimental groups. Similar results were obtained in mice in which LRP was inhibited via overexpression of the RAP gene in liver by adenoviral gene transduction. In contrast, administration of mannan, a mannose receptor antagonist, resulted in identical clearances (0.22 mL/min in controls and 0.24 mL/min in EE-treated mice). Northern blot analysis showed a 6-fold increase in mannose receptor mRNA expression in the nonparenchymal liver cells of EE-treated mice, whereas the parenchymal LRP mRNA levels remained unchanged. These findings were confirmed at the protein level by ligand blotting and Western blotting analysis. Our results demonstrate that EE treatment results in increased plasma clearance rate of t-PA via induction of the mannose receptor and could explain for the inverse relationship between estrogen status and plasma t-PA concentrations as observed in humans.
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PMID:Increased clearance explains lower plasma levels of tissue-type plasminogen activator by estradiol: evidence for potently enhanced mannose receptor expression in mice. 1043 21

The clearance and degradation of extracellular A beta is critical for regulating beta-amyloid deposition, a major hallmark of brains of patients with A beta in Alzheimer's Disease. The low-density lipoprotein receptor-related protein, LRP1, is a large endocytic receptor that significantly contributes to the balance between degradation and production of A beta. An extracellular portion of the LRP, known as the cluster II region can bind to the secreted form of APP (sAPP-KPI). We show here that a GST fusion protein containing the cluster II region of LRP can be used as a 'mini-receptor' that specifically binds to sAPP-KPI from conditioned cultured medium. The binding between the GST-LRP-cluster II fusion protein and sAPP-KPI can be inhibited with the strong binding ligand of LRP1, called receptor-associated protein (RAP). Furthermore, a cell-based in vitro assay system has been developed to monitor the production of total A beta and A beta(1-42) in the presence and absence of RAP in Chinese hamster ovary (CHO) cell lines both deficient in LRP and expressing LRP. A 3-day treatment of the L2 (CHO cells deficient in LRP and overexpressing APP751) and L3 (CHO cells expressing LRP and overexpressing APP751) cell lines with RAP showed a decrease in total A beta and, interestingly, also a decrease in the ratio of A beta42/A beta(total). This cell-based model system and LRP-cluster II mini-receptor will be very useful for screening novel compounds that can reduce A beta accumulation by inhibiting binding of APP-KPI to LRP1.
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PMID:The role of the low-density lipoprotein receptor-related protein (LRP1) in Alzheimer's A beta generation: development of a cell-based model system. 1221 91

In this study, we demonstrate that B-type natriuretic peptide (BNP) opposed angiotensin II (Ang II)-stimulated de novo cholesterol biosynthesis, cellular cholesterol uptake, cholesterol transfer to the inner mitochondrial membrane, and steroidogenesis, which are required for biosynthesis of steroid hormones such as aldosterone and cortisol in primary human adrenocortical cells. BNP dose-dependently stimulated intracellular cGMP production with an EC(50) of 11 nm, implying that human adrenocortical cells express the guanylyl cyclase A receptor. cDNA microarray and real-time RT-PCR analyses revealed that BNP inhibited Ang II-stimulated genes related to cholesterol biosynthesis (acetoacetyl coenzyme A thiolase, HMG coenzyme A synthase 1, HMG coenzyme A reductase, isopentenyl-diphosphate Delta-isomerase, lanosterol synthase, sterol-4C-methyl oxidase, and emopamil binding protein/sterol isomerase), cholesterol uptake from circulating lipoproteins (scavenger receptor class B type I and low-density lipoprotein receptor), cholesterol transfer to the inner mitochondrial membrane (steroidogenic acute regulatory protein), and steroidogenesis (ferredoxin 1,3beta-hydroxysteroid dehydrogenase, glutathione transferase A3, CYP19A1, CYP11B1, and CYP11B2). Consistent with the microarray and real-time PCR results, BNP also blocked Ang II-induced binding of (125)I-labeled low-density lipoprotein and (125)I-labeled high-density lipoprotein to human adrenocortical cells. Furthermore, BNP markedly inhibited Ang II-stimulated release of estradiol, aldosterone, and cortisol from cultured primary human adrenocortical cells. These findings demonstrate that BNP opposes Ang II-induced steroidogenesis via multiple steps from cholesterol supply and transfer to the final formation of steroid hormones. This study provides new insights into the cellular mechanisms by which BNP modulates Ang II-induced steroidogenesis in the adrenal gland.
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PMID:B-Type natriuretic peptide inhibited angiotensin II-stimulated cholesterol biosynthesis, cholesterol transfer, and steroidogenesis in primary human adrenocortical cells. 1747 52

Puerarin (PU), a natural flavonoid, has been reported to have many benefits and medicinal properties. The aim of the present study was to investigate the effects of puerarin on hepatic oxidative stress and hyperlipidemia in rats exposed to lead. Our data showed that puerarin significantly prevented lead-induced hepatotoxicity, indicated by both diagnostic indicators of liver damage (serum aminotransferase levels) and histopathological analysis. Moreover, lead-induced profound elevation of ROS production and oxidative stress, as evidenced by increasing of lipid peroxidation level, reducing of GPx, GST, GR and GCL activities and depleting of intracellular reduced GSH level in liver, were suppressed by treatment with puerarin. Furthermore, the increase of serum cholesterol, triglycerides and LDL induced by lead was effectively suppressed by puerarin. The HDL level in the lead treatment rats was also increased by puerarin. Western blot analysis showed that puerarin remarkably inhibited hyperlipidemia by regulating the expression of cholesterol 7a-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and low-density lipoprotein receptor (LDL-R) in liver of lead treated rats. Altogether, these results suggest that puerarin could protect the lead-induced liver injury and hyperlipidemia by reducing ROS production, renewing the activities of antioxidant enzymes and influencing expression of hepatic lipid biosynthesis and metabolism genes.
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PMID:Protective role of puerarin on lead-induced alterations of the hepatic glutathione antioxidant system and hyperlipidemia in rats. 2200 Nov 70


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