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)

A putative dihydrofolate reductase (DHFR) module has been identified in neuronal nitric oxide synthase, consisting of amino acids 558-721, and is proposed to be the site of tetrahydrobiopterin (BH4) binding. This polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1. The protein binds N omega-nitro-L-arginine (NNA) tightly, but this binding is not stimulated by BH4. cDNAs for Module II (residues 220-557) and Module III (residues 220-721) have been expressed as fusion proteins with GST. Module II does not bind NNA. However, Module III does bind NNA and binding is significantly stimulated by BH4. These observations are taken as strong evidence that the DHFR module contains the L-arginine binding site and, presumably, the BH4 binding site by analogy to its homology with DHFR, but that tight binding of BH4 requires amino acids 220-577.
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PMID:Modular structure of neuronal nitric oxide synthase: localization of the arginine binding site and modulation by pterin. 753 58

cDNA clones encoding proteins related to the aggrecan/versican family of proteoglycan core proteins have been isolated with antisera against rat brain synaptic junctions. Two sets of overlapping cDNAs have been characterized that differ in their 3'-terminal regions. Northern analyses with probes derived from unique regions of each set were found to hybridize with two brain-specific transcripts of 3.3 and 3.6 kilobases (kb). The 3.6-kb transcript encodes a polypeptide that exhibits 82% sequence identity with bovine brevican and is thought to be the rat ortholog of brevican. Interestingly, the polypeptide deduced from the open reading frame of the 3.3-kb transcript is truncated just carboxyl-terminal of the central domain of brevican and instead contains a putative glypiation signal. Antibodies raised against a bacterially expressed glutathione S-transferase-brevican fusion protein have been used to show that both soluble and membrane-bound brevican isoforms exist. Treatment of the crude membrane fraction and purified synaptic plasma membranes with phosphatidylinositol-specific phospholipase C revealed that isoforms of brevican are indeed glycosylphosphatidylinositol-anchored to the plasma membrane. Moreover, digestions with chondroitinase ABC have indicated that rat brevican, like its bovine ortholog, is a conditional chondroitin sulfate proteoglycan. Immunohistochemical studies have shown that brevican is widely distributed in the brain and is localized extracellularly. During postnatal development, amounts of both soluble and phosphatidylinositol-specific phospholipase C-sensitive isoforms increase, suggesting a role for brevican in the terminally differentiating and the adult nervous system.
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PMID:Brevican, a chondroitin sulfate proteoglycan of rat brain, occurs as secreted and cell surface glycosylphosphatidylinositol-anchored isoforms. 759 78

Molecular cloning of the structural gene for adenylate cyclase (cya) of the cyanobacterium Anabaena cylindrica was carried out by complementation of an Escherichia coli strain defective in the cya gene. The cya-defective strain produced significant amounts of cyclic AMP when it was transformed with the cya gene isolated from A. cylindrica. This gene encodes a polypeptide consisting of 502 amino acid residues (molecular weight, 55,300). The deduced primary protein structure showed that the carboxyl-terminal region of the adenylate cyclase of A. cylindrica shows strong structural similarity to the conserved regions of the adenylate cyclases of various eukaryotes. No similarity was found between the amino acid sequences of the cya gene of A. cylindrica and that of E. coli. A hydropathy plot suggests that this protein has two hydrophobic regions, a transmembrane span and a signal peptide. An antiserum specific to this adenylate cyclase was prepared by immunizing a rabbit with a glutathione S-transferase-adenylate cyclase fusion protein expressed in E. coli. This antiserum recognized a 55-kDa protein in Anabaena cell lysates. Subcellular fractionation analysis showed that A. cylindrica adenylate cyclase localized in the thylakoid membrane.
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PMID:Molecular cloning of the cyanobacterial adenylate cyclase gene from the filamentous cyanobacterium Anabaena cylindrica. 766 7

p120 GTPase-activating protein (GAP) is a negative regulator of Ras that functions at a key relay point in signal transduction pathways that control cell proliferation. Among other proteins, p120 GAP associates with p190, a GAP for the Ras-related protein, Rho. To characterize the p120.p190 interaction further, we used bacterially expressed glutathione S-transferase fusion polypeptides to map the regions of p120 necessary for its interactions with p190. Our results show that both the N-terminal and the C-terminal SH2 domains of p120 are individually capable of binding p190 expressed in a baculovirus/insect cell system. Moreover, the two SH2 domains together on one polypeptide bind synergistically to p190, and this interaction is dependent on tyrosine phosphorylation of p190. In addition, mutation of the highly conserved Arg residues in the critical FLVR sequences of both SH2 domains of full-length p120 reduces binding to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120.p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120.p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.
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PMID:Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein. 762 1

We report here the first characterization of a gene encoding a homogentisate dioxygenase, the Aspergillus nidulans hmgA gene. The HmgA protein catalyzes an essential step in phenylalanine catabolism, and disruption of the gene results in accumulation of homogentisate in broths containing phenylalanine. hmgA putatively encodes a 448-residue polypeptide (Mr = 50,168) containing 21 histidine and 23 tyrosine residues. This polypeptide has been expressed in Escherichia coli as a fusion to glutathione S-transferase, and the affinity-purified protein has homogentisate dioxygenase activity. A. nidulans, an ascomycete amenable to classical and reverse genetic analysis, is a good metabolic model to study inborn errors in human Phe catabolism. One such disease, alkaptonuria, was the first human inborn error recognized (Garrod, A. E. (1902) Lancet 2, 1616-1620) and results from loss of homogentisate dioxygenase. Here we take advantage of the high degree of conservation between the amino acid sequences of the fungal and higher eukaryote enzymes of this pathway to identify expressed sequence tags encoding human and plant homologues of HmgA. This is a significant advance in characterizing the genetic defect(s) of alkaptonuria and illustrates the usefulness of our fungal model.
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PMID:Molecular characterization of a gene encoding a homogentisate dioxygenase from Aspergillus nidulans and identification of its human and plant homologues. 767 53

A lambda gt11 Fasciola hepatica cDNA library was previously constructed from poly(A)+ RNA extracted from adult worms. A cDNA encoding F. hepatica glutathione S-transferase (FhGST) (GenBank Accession Number M93434) was cloned by screening the library with a rabbit anti-FhGST antiserum. The FhGSTs are suspected to be early developmentally expressed-proteins that act as potent immunogens and may be useful as candidate vaccines against the parasite. The cDNA was sequenced and contains 627 translated bases encoding a 24,211-Dalton polypeptide with 209 amino acids and an isoelectric point (pI) of 5.324. This polypeptide has significant homology with 26-kD GSTs from Schistosoma mansoni (57.42%) and S. japonicum (57.14%). In addition, we have identified the predicted T cell epitopes in the FhGST protein molecule.
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PMID:Sequence analysis of a Fasciola hepatica glutathione S-transferase cDNA clone. 768 83

A mu-class glutathione S-transferase (GST) cDNA clone, pHMB1, from rabbit liver has been constructed, using a 748-base-pair fragment of GST Yb1 cDNA as a probe. The nucleotide sequence of pHMB1 has been determined, and the complete amino acid sequence has been deduced. Recombinant clone pHMB1 contains a cDNA insert of 1443 base pairs with 654 nucleotides of open reading frame, 33 nucleotides of 5'-untranslated region, and 756 nucleotides of 3'-untranslated region. The open reading frame encodes a polypeptide (rbGST mu I) comprising 218 amino acids with molecular weight of 25,417. Compared to published mu-class GST sequences, rbGST mu I is 73 and 77% identical to rat Yb1 and human GST4 in amino acid sequence, respectively. The pHMB1 was expressed in Escherichia coli using expression vector pIH821 and the expressed GST was purified as a single band on polyacrylamide gel electrophoresis by maltose- and glutathione-affinity column chromatography. Rabbit liver GST protein expressed by this system was catalytically active. The functional characterization was done on the expressed protein. The rabbit liver GST expressed in E. coli showed greater activity toward 1,2-dichloro-4-nitrobenzene than mu-class isozymes in rabbit hepatic tissue (T. Primiano and R.F. Novak (1993) Arch. Biochem. Biophys. 301, 404-410). Enzymatic activity of expressed protein toward the substrate 1-chloro-2,4-dinitrobenzene was inhibited by triethyltin bromide, Cibacron blue, triphenyltin chloride, bromosulfophthalein, and hematin. RNA blot hybridization demonstrated that the pHMB1 mRNA was well expressed in rabbit liver, brain, and kidney.
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PMID:Cloning and expression of a cDNA for mu-class glutathione S-transferase from rabbit liver. 773 73

Cytoplasmic dynein and ncd, a kinesin-related protein from Drosophila, are motor proteins that move toward the minus ends of microtubules, while kinesin moves to the microtubule plus end. In previous work, we examined the nucleotide dependence of motility and enzymatic activity by kinesin [Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F., & Vale, R. D., (1991) J. Cell Biol. 112, 1189-1197]. In this study, we examined these activities of the cytoplasmic dynein from bovine brain and ncd in order to explore what enzymatic features might be shared by these two minus-end-directed motors. Both ncd and cytoplasmic dynein demonstrated an activation of ATPase activity upon the addition of microtubules (30-fold and 6-fold, respectively). A significant difference between ncd and cytoplasmic dynein was their relative sensitivity to vanadate and to aluminum fluoride. In contrast to cytoplasmic dynein, ncd polypeptide was not cleaved by UV-vanadate treatment, and its ATPase and motility were unaffected by vanadate (up to 0.1 mM). When the nucleotide requirement for movement as examined using a battery of 20 nucleotides and nucleotide analogues, cytoplasmic dynein was found to exhibit a specificity very similar to that of axonemal dyneins from Tetrahymena. Surprisingly, however, the nucleotide specificities of in vitro motility produced by ncd or its construct, GST/MC1 (a fusion protein of glutathione S-transferase and 210-700 of the predicted ncd amino acid sequence), were quite distinct from that of kinesin. Thus, the nucleotide specificity profiles of members of the kinesin motor superfamily do not appear to be identical.
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PMID:Comparison of the motile and enzymatic properties of two microtubule minus-end-directed motors, ncd and cytoplasmic dynein. 784 16

The heat-induced expression of heat shock proteins, called the cellular stress response, is mediated by heat shock transcription factor 1 (HSF1). HSF1 exists in unstressed cells in an inactive form, which is converted to the DNA binding from upon exposure of cells to elevated temperature. We have developed a protocol for isolation of the non-DNA binding form of recombinant mouse HSF1, involving expression and affinity purification of HSF1 as a fusion with the glutathione S-transferase protein in Escherichia coli, followed by specific protease cleavage to release pure HSF1 protein. We report here that the purified inactive HSF1 can be converted to the DNA binding form by heat treatment in vitro. Chemical cross-linking analysis demonstrates that this conversion is accompanied by oligomerization of HSF1 from a monomeric to a trimeric native structure, similar to that observed for HSF1 in heat-shocked cells. These results indicate that elements residing in the HSF1 polypeptide are sufficient both for maintenance of this factor in the non-DNA binding from and for its heat-induced conversion to the DNA binding form and support a role for HSF1 as the "molecular thermostat" in eukaryotic cells, which senses adverse environmental conditions and activates the cellular stress response.
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PMID:Heat-inducible DNA binding of purified heat shock transcription factor 1. 785 5

To determine the minimal sequence requirements for steroid binding and dimerization of human sex hormone-binding globulin (SHBG), the SHBG polypeptide and various SHBG deletion mutants were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. Fusion proteins containing the complete SHBG sequence, or the first 177 N-terminal residues of SHBG, bound steroids with high affinity and specificity. Further deletions from the C-terminus severely compromised steroid-binding activity, as did N-terminal deletions beyond residue 18 in the SHBG sequence. Thus, residues 18-177 in SHBG encompass a region required for its steroid-binding activity, and a disulfide bridge normally present between Cys-164 and Cys-188 in SHBG is not obviously essential for steroid binding. Most of the GST/SHBG fusion proteins undergo cleavage at 4 degrees C, releasing immunoreactive polypeptides that correspond approximately in size to their respective SHBG sequences. The 23-kDa immunoreactive cleavage product released from the fusion protein containing residues 1-205 in the SHBG sequence (SHBG 1-205) has a 50-fold greater steroid-binding capacity but a 7.5-fold lower affinity than its parent fusion protein. In addition, the 22-kDa immunoreactive polypeptide released from SHBG(1-194) binds steroid, and its dimerization is promoted by steroid ligands that bind SHBG with high affinity. These data suggest that the N-terminal region of SHBG dimerizes readily in the absence of GST and in doing so acquires steroid-binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Resolution of the steroid-binding and dimerization domains of human sex hormone-binding globulin by expression in Escherichia coli. 788 Aug 17


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