Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An alternative enzyme linked immunosorbent assay (ELISA) system was developed to analyze antibodies to human papillomavirus capsid antigens. The assay uses glutathione crosslinked to casein to capture the major capsid protein L1 from human papillomavirus (HPV) types 6b, 16 and 18 fused to glutathione S-transferase (GST) as antigen. The method allows efficient one-step purification of L1 fusion protein from crude bacterial lysates on ELISA plates coated with glutathione casein. The GST-L1 capture ELISA detected HPV 16 antibodies with high type specificity. Comparison with the current "gold-standard" for L1-serology that uses virus-like particles (VLP) as antigen demonstrated similar assay sensitivity. Pairwise comparison of the absorbance values of 105 human sera obtained in the two ELISA formats for HPV 16 showed a R(2) value of linear regression of 0.68. Conformity of the two ELISAs in classification of sera as HPV 16 L1 antibody-positive or -negative was verified with Cohen's kappa test, yielding a value of 0.62. These data indicate that the GST-L1 capture ELISA is similar in performance to the VLP ELISA. The ease of antigen production and purification in the GST-based ELISA will be advantageous to screen large sample numbers in vaccine trials or epidemiological studies examining immune responses to many HPV types in parallel.
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PMID:HPV antibody detection by ELISA with capsid protein L1 fused to glutathione S-transferase. 1236 30

Cell surface receptor membrane localization is strongly dependent on protein-protein interactions often involving regulation by phosphorylation/dephosphorylation of the intracellular domains of membrane proteins. The present study was carried out to identify metabotropic glutamate receptor (mGluR) 3 regulatory binding proteins. Using the yeast two-hybrid technique, we found that the 50-aa C-terminal cytoplasmic tail of mGluR3 interacts specifically with protein phosphatase 2Calpha (PP2Calpha). This interaction was confirmed by GST pull-down and coimmunoprecipitation assays. mGluR3 interacts with PP2Calpha, beta, gamma, and delta isoforms; however, among the mGluR family only mGluR3 interacted with PP2C. The minimal interacting domain of mGluR3 comprised residues 836-855. Alignment between mGluR3 and mGluR2, a closely related group II receptor, indicated that this domain is not conserved between the two receptors. The mGluR3 cytoplasmic C-terminal tail contains one phosphorylation site for protein kinase A (Ser-845), but the phosphatase that dephosphorylates this site has not been previously identified. We find that PP2C, but not PP1, PP2A, or PP2B, dephosphorylates the mGluR3 cytoplasmic tail in vitro. The dephosphorylated form of the mGluR3 cytoplasmic tail, but not the equivalent region of mGluR2, inhibited PP2C assayed by using [32P]casein as a substrate. However, phosphorylation of the mGluR3 cytoplasmic tail at Ser-845 inhibits the interaction with PP2C. These results indicate distinct functions for mGluR2 and mGluR3 and suggest a dynamic regulation of mGluR3 by PP2C.
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PMID:Protein phosphatase 2C binds selectively to and dephosphorylates metabotropic glutamate receptor 3. 1466 50

Proteases play a pivotal role in epidermal differentiation and desquamation. Separation of a total protein extract from human reconstructed epidermis by two-dimensional gel electrophoresis and subsequent peptide analysis of a specific protein spot identified a new protein exhibiting similarities with the retroviral aspartic protease family. Cloning of the corresponding full-length cDNA revealed an open reading frame encoding for a new protease of 343 amino acids, containing a putative aspartic protease catalytic domain. We named this protein Skin ASpartic Protease (SASPase). RT-PCR and northern blot analysis of various human tissues revealed that SASPase was specifically expressed within the epidermis. Immunohistochemical analysis showed a particularly intense expression restricted to the granular layers, whereas in diseased skin, its expression was changed. Western blot analysis, using a monoclonal antibody, revealed the expression of two forms of the enzyme: a 28 kDa putative proform and the active 14 kDa form. Recombinant truncated SASPase (SASP28) was generated from a prokaryotic expression system in Escherichia coli as a fusion protein with GST. SASP28 degraded insulin and to a lesser extent casein with a pH optimum of 5. As seen for retroviral proteases, an auto-activation processing was evidenced, generating a 14 kDa protein (SASP14). Site-directed mutagenesis inhibited auto-activation of the enzyme. Indinavir, a potent HIV protease inhibitor used in AIDS therapy, had a significant inhibitory effect on rSASPase auto-activation, which could explain its side effects on skin.
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PMID:Identification and characterization of a novel retroviral-like aspartic protease specifically expressed in human epidermis. 1609 38

Phototropin is a blue light photoreceptor for tropic responses, relocation of chloroplasts, and stomata opening in plants. Phototropin has two chromophoric domains named light-oxygen-voltage-sensing (LOV) 1 and 2 in the N-terminal half, and a serine/threonine (Ser/Thr) protein kinase motif in the C-terminal half. Concerning the kinase activity of phototropin, only autophosphorylation has been detected so far. However, we found that phototropin can phosphorylate a protein other than phototropin itself. Bacterially expressed Arabidopsis phototropin 2 kinase domain (KD) with GST-tag showed a constitutive kinase activity on casein, a common in vitro substrate of Ser/Thr protein kinase. By using this in vitro assay system, the roles of each LOV domain were studied. Addition of LOV2 to KD (GST-L2-KD) inhibits the kinase activity that is canceled by light. This light activation of kinase disappeared on introduction of a mutation blocking photochemical reaction in the LOV2 domain. Accordingly, LOV2 domain acts as a major light-regulated molecular switch of casein phosphorylation. Interestingly, isolated LOV2 from the KD still binds to the KD in a light-dependent manner and functions in similar ways, indicating the role of LOV2 domain as an inhibitor of the kinase activity in the substrate phosphorylation. LOV1, in contrast, contributes little to the photoactivation in GST-L1-L2-KD; however, it acts as an attenuator of the light activation of the kinase by LOV2.
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PMID:Blue light-regulated molecular switch of Ser/Thr kinase in phototropin. 1615 Jul 10

Members of the Casein Kinase 1 (CK1) family are implicated in the regulation of a variety of physiological processes like development and circadian rhythm, as well as in diseases like cancer and Alzheimer's disease. From that perspective, CK1 family members are interesting targets for potential chemotherapy. We describe here a rapid and efficient method for the purification of CK1 by affinity chromatography on an immobilised fragment of axin. Axin is a scaffolding protein that interacts with a multitude of proteins, amongst them APC, GSK-3, beta-catenin, CK1alpha, delta, and epsilon, and PP2A. A GST-tagged axin peptide (residues 495-684) was produced in Escherichia coli and either immobilised on glutathione agarose beads or purified and immobilised on CNBr-activated sepharose 4B. These "GST-axin" matrices were found to selectively bind native CK1alpha and CK1epsilon from porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to efficiently purify large amounts of active native CK1 for screening purposes. This single step purification method also provides a convenient tool to follow the status of the axin-binding CK1 isoforms alpha, delta, and epsilon (protein levels, composition of isoforms, kinase activity) under different physiological settings.
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PMID:Purification of CK1 by affinity chromatography on immobilised axin. 1743 49

Polychlorinated biphenyls (PCBs) are ubiquitious lipophilic environmental pollutants. Some of the PCB congeners and mixtures of congeners have tumor promoting activity in rat liver. The mechanism of their activity is not fully understood and is likely to be multifactorial. The aim of this study was to investigate if the resident liver macrophages, Kupffer cells, are important in the promoting activity of PCBs. The hypothesis of this study was that the inhibition of Kupffer cell activity would inhibit hepatic tumor promotion by PCBs in rats. To test our hypothesis, we studied the effects of Kupffer cell inhibition by dietary glycine (an inhibitor of Kupffer cell secretory activity) in a rat two-stage hepatocarcinogenesis model using 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153, a non-dioxin-like PCB) or 3,3',4,4'-tetrachlorobiphenyl (PCB-77, a dioxin-like PCB) as promoters. Diethylnitrosamine (DEN, 150 mg/kg) was administered to female Sprague-Dawley rats, which were then placed on an unrefined diet containing 5% glycine (or casein as nitrogen control) starting two weeks after DEN administration. On the third day after starting the diets, rats received PCB-77 (300 micromol/kg), PCB-153 (300 micromol/kg), or corn oil by i.p. injection. The rats received a total of 4 PCB injections, administered every 14 days. The rats were euthanized on the 10th day after the last PCB injection, and the formation of altered hepatic foci expressing placental glutathione S-transferase (PGST) and the rate of DNA synthesis in these foci and in the normal liver tissue were determined. Glycine did not significantly affect foci number or volume. PCB-153 did not significantly increase the focal volume, but increased the number of foci per liver, but only in the rats not fed glycine; PCB-77 increased both the foci number and their volume in both glycine-fed and control rats. Glycine did not alter the PCB content of the liver, but did increase the activity of 7-benzyloxyresorufin O-dealkylase (BROD) in liver microsomes from PCB-153 treated rats. However, glycine did not affect the induction of ethoxyresorufin O-dealkylase activity by PCB-77 in liver microsomes. Glycine diminished hepatocyte proliferation in PGST-positive foci, but not in normal tissue. Overall these results do not support the hypothesis that dietary glycine inhibits the promoting activities of PCBs. The observations that PCB-153 increased the number of foci per liver in control rats but not glycine-fed rats and that dietary glycine reduced cell proliferation in PGST-positive foci, however, do not allow us to completely rule out a role for dietary glycine. But the data overall indicate that Kupffer cells likely do not contribute to the tumor promoting activities of PCB-77 and PCB-153.
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PMID:The effect of dietary glycine on the hepatic tumor promoting activity of polychlorinated biphenyls (PCBs) in rats. 1770 65

The most essential and crucial step during the pathogenesis of transmissible spongiform encephalopathy is the conformational change of cellular prion protein to pathologic isoform. Casein kinase II (CK2) is a ubiquitously expressed and evolutionarily conserved pleiotropic protein kinase that is essential for viability. To explore the possible molecular interaction between CK2 and prion protein (PrP), the full-length sequences of human CK2alpha and CK2beta complementary DNA were amplified with reverse transcription-polymerase chain reaction using the total messenger RNA from cell line SH-SY5Y as the template; then, the fusion proteins histidine-CK2alpha and glutathione S-transferase-histidine-CK2beta were expressed in Escherichia coli. The interaction between CK2 and PrP was evaluated with co-immunoprecipitation and pull-down assays. The results demonstrated that recombinant PrP bound specifically with CK2alpha, but not with CK2beta. The native CK2 and PrP in hamster brains interacted with each other, forming protein complexes. Three different glycosylated forms of PrP (diglycosylated, monoglycosylated and unglycosylated PrP) from normal brains interacted with the CK2alpha subunit, though the unglycosylated PrP seemed to have a stronger binding ability with CK2alpha subunit. The domain responsible for interacting with CK2alpha was located at the C-terminal segment of PrP (residues 91-231). This study proposed reliable experimental data for the molecular interaction between PrP and CK2alpha (both in recombinant and native categories), scientific clues for further assessing the potential biological significance of the PrP-CK2 interaction, and the possible role of CK2 in the pathogenesis of prion diseases.
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PMID:Casein kinase II interacts with prion protein in vitro and forms complex with native prion protein in vivo. 1908 2

A cDNA highly homologous to the known catalytic alpha subunit of protein kinase CK2 was cloned from maize (Zea mays). It was designated ZmCK2alpha-4 (accession no. AAF76187). Sequence analysis shows that ZmCK2alpha-4 and the previously identified ZmCK2alpha-1 (accession no. X61387) are transcribed from the same gene, ZmPKCK2AL (accession no. Y11649), but at different levels in various maize organs and at different stages of development. The cDNA encoding ZmCK2alpha-4 has three potential translation initiation sites. The three putative variants of ZmCK2alpha-4 were expressed in Escherichia coli as GST-fusion proteins and purified from bacterial extracts. In contrast to the previously characterized ZmCK2alphas, the obtained GST:ZmCK2alpha-4 proteins were catalytically inactive as monomers or in the presence of equimolar amounts of the human CK2beta. However, GST:ZmCK2alpha-4 did phosphorylate casein in the presence of a large excess of the beta subunit. The activity of ZmCK2alpha-4 toward casein could also be stimulated by increasing ATP concentration. Modeling studies have shown that there is no interaction between the N-terminal segment of ZmCK2alpha-4 and the activation loop responsible for constitutive catalytic activity of CK2alpha. Preliminary results suggest that ZmCK2alpha-4 may function as a negative regulator of other CK2s, and at certain circumstances as a holoenzyme which catalytic activity is stimulated by specific regulatory subunit(s).
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PMID:A novel splicing variant encoding putative catalytic alpha subunit of maize protein kinase CK2. 1947 94

The consumption of soy and soy isoflavones has been associated with a decreased risk of certain cancers. A factor contributing to this dietary chemoprevention is the activity of phase I and II biotransformation enzymes. This study evaluated the hypothesis that dietary soy isoflavones will increase hepatic and extrahepatic quinone reductase (QR), UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) phase II enzyme activities, under short-term feeding and basal (non-pharmacologic-induced) conditions. Male and female Swiss Webster mice were fed for 1, 3, 5, or 7 days of one of four treatments: control (casein AIN-93G) or control supplemented with flavone (positive control), genistein, or daidzein aglycones at 1,500 mg/kg of diet. QR activity was increased by daidzein in the liver, by both isoflavones in the kidney and small intestine, and by genistein in the heart. Genistein and daidzein slightly decreased UGT activities in some tissues. Liver GST activity was decreased by genistein in females. In contrast, genistein and daidzein increased kidney GST activity. In general, the greatest effects of isoflavones on phase II enzymes were observed in liver and kidney tissues, occurring at day 3, and peaking at day 5. Sex effects in the liver and kidney included females exhibiting higher QR activities and males exhibiting higher UGT and GST activities. In conclusion, individual soy isoflavones modulate phase II enzymes in mice under short-term feeding and basal conditions. This study provides insights into the actions of isolated isoflavones in mice.
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PMID:Regulation of phase II enzymes by genistein and daidzein in male and female Swiss Webster mice. 2004 75

Enzyme-linked immunosorbent assays (ELISA) have been widely used to determine quantitatively autoantibodies. However, the processes for the purification and immobilization of antigens in conventional ELISA methods include multiple steps, which have hampered the application for screening of autoantibodies. Here, we have developed a novel ELISA system using the plates pre-coated with glutathione casein to capture recombinant proteins fused to N-terminal glutathione S-transferase (GST). The GST-fused proteins were synthesized with the wheat germ cell-free protein production system. Thus, the present system combined the GST-capture ELISA with the cell-free protein production system, which allowed immobilization of the recombinant proteins with one-step purification. Using this ELISA method, we determined whether rheumatoid factors (RF), which have been considered as one of the representative disease-specific autoantibodies for rheumatoid arthritis (RA), were genetically associated with severity of arthritis in a mouse model for RA, MRL/Mp-lpr/lpr (MRL/lpr). GST-fused human IgG1-Fc (GST-Fc), synthesized with the robotic protein synthesizer, were used as reactants for RF. Serum samples for RF were prepared from 11 lines of a recombinant inbred mouse strain, MXH/lpr, which was established from intercrosses between MRL/lpr and non-arthritic C3H/HeJ-lpr/lpr (C3H/lpr) strains, composed of a different genomic recombination derived from the parental strains in each line. A correlation of RF titers with the severity of the arthritis in these lines was not significant, indicating genetic dissociation of RF from arthritis and that RF is not necessarily required for the development of RA. The present method may provide high-throughput screening for determining the disease-specific autoantibodies in autoimmune diseases.
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PMID:Evaluating the role of rheumatoid factors for the development of rheumatoid arthritis in a mouse model with a newly established ELISA system. 2020 15


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