Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Trypanothione reductase (TR) is a target for drug design since it is unique to trypanosomatids, substituting for the otherwise ubiquitous enzyme, glutathione reductase. We report the cloning and sequencing of several cDNAs and genes encoding Crithidia fasciculata TR, the structure of which has recently been solved by crystallography. Single base polymorphisms are detected in cDNAs (containing 80% of the coding sequence) and two different genomic clones, including a glutamine to glutamate change in the C-terminal region of the TR coding region; other nucleotide changes are silent. Homology (from genomic clones, both of which contained signals appropriate for expression) to the Trypanosoma congolense gene was 63% at the nucleic acid level, with 68% amino acid identity; the significance of homologies to human and Escherichia coli glutathione reductase sequences is discussed. Polymorphic sites in the genomic clones included sites found in the cDNAs, indicating that differences existing in the genomic sequence are real, and propagated to RNA.
Mol Biochem Parasitol 1992 Jan
PMID:Cloning, sequencing, and demonstration of polymorphism in trypanothione reductase from Crithidia fasciculata. 154 16

The Corynebacterium glutamicum gdh gene encoding NADP-dependent glutamate dehydrogenase (GDH) has been isolated by complementation of the Escherichia coli gdh mutant PA340. The gdh gene was subcloned into the E. coli/C. glutamicum shuttle vector pEK0 and introduced into C. glutamicum. Recombinant strains showed approximately eightfold higher specific GDH activity (15U mg protein-1) relative to the wild type (1.8U mg protein-1). Physiological studies with wild-type and recombinant C. glutamicum strains revealed no indication of significant regulation of gdh expression. The DNA sequence of 2082 bp, including the gdh gene, 5'-, and 3'-flanking regions, was determined. The structural gene consists of 1344 bp and codes for a polypeptide of 448 amino acid residues (Mr 49,152) showing up to 53.6% identity with reported amino acid sequences of glutamate dehydrogenases from other organisms. Northern blot hybridization revealed a 1.65kb mRNA transcript, indicating that the gdh gene of C. glutamicum is monocistronic. Transcription occurred from a G residue located 284 bp upstream of the AUG considered to be the translational initiation codon.
Mol Microbiol 1992 Feb
PMID:Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase. 155 46

In a recent paper we described a system in which glucocorticoid receptors associate with particulate complexes containing tubulin [Cancer Res. 49 (1989) 2222s-2229s]. When L cell cytosol is mixed with a microtubule stabilizing buffer and heated to 37 degrees C, the receptor becomes associated with a complex that can be centrifuged out of solution at 150,000 g. In this work we show that the glucocorticoid receptor-cytoskeletal protein complex forms in a temperature and glutamate-dependent manner. Molybdate does not affect generation of the cytoskeletal protein complex but it inhibits association of the receptor with the complex. This suggests that transformation of the receptor to its DNA-binding form is required for interaction with the cytoskeletal complex. Colchicine has no effect on generation of the particulate complex or on the association of receptor with it, suggesting that formation of the complex does not represent a classic in vitro process of tubulin polymerization.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Association of the transformed glucocorticoid receptor with a cytoskeletal protein complex. 156 45

5-Lipoxygenase-activating protein (FLAP) is specifically labeled by [125I]L-669,083 and [125I]L-691,678, photoaffinity analogues of two classes of potent leukotriene biosynthesis inhibitors. Because human FLAP contains only a single tryptophan residue at position 72 and two internal methionine residues at positions 89 and 125, we have used reagents that specifically cleave at these residues, in conjunction with antipeptide antisera, to localize the site of attachment of the photoaffinity ligands. Immunoprecipitation of specifically labeled peptide fragments after digestion of photoaffinity-labeled FLAP by iodosobenzoic acid at 72Trp demonstrates that the inhibitors bind to FLAP amino-terminal to this residue. This finding is consistent with similar immunoprecipitation studies after digestion at methionine residues using cyanogen bromide. These findings localize the site of attachment of the inhibitors to a region of FLAP that includes the hydrophilic loop between the proposed first and second transmembrane regions. Based on these findings, site-directed mutagenesis of human FLAP was performed to define key amino acids involved in inhibitor binding. Using a radioligand binding assay, analysis of mutants of human FLAP expressed in COS-7 cells demonstrates that a number of residues in the amino-terminal half of the first hydrophilic loop of the protein can be deleted without significantly affecting inhibitor binding. In contrast, no inhibitor binding was detectable with mutants in which amino acid residues in the carboxyl-terminal half of this loop were deleted. Furthermore, a point mutation of 62Asp to asparagine results in a mutant with dramatically reduced affinity for inhibitors. This loss of affinity was not displayed by a mutant in which 62Asp was mutated to a glutamate residue, suggesting that a negative charge associated with residue 62 may be critical for inhibitor binding. The roles that amino acid residues in the carboxyl-terminal half of the first hydrophilic loop of FLAP may play in the binding of leukotriene biosynthesis inhibitors are currently under investigation.
Mol Pharmacol 1992 Jul
PMID:Identification of amino acid residues of 5-lipoxygenase-activating protein essential for the binding of leukotriene biosynthesis inhibitors. 163 56

The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.
J Mol Biol 1991 Aug 05
PMID:Molecular structure of cytochrome c2 isolated from Rhodobacter capsulatus determined at 2.5 A resolution. 165 96

We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.
Mol Cell Biol 1991 Sep
PMID:Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae. 165 57

The N-methyl-D-aspartate (NMDA) receptor is believed to play a major role in learning and in excitotoxic neuronal damage associated with stroke and epilepsy. Pregnenolone sulfate, a neurosteroid, specifically enhances NMDA-gated currents in spinal cord neurons, while inhibiting receptors for the inhibitory amino acids glycine and gamma-aminobutyric acid, as well as non-NMDA glutamate receptors. This observation is consistent with the hypothesis that neurosteroids such as pregnenolone sulfate are involved in regulating the balance between excitation and inhibition in the central nervous system.
Mol Pharmacol 1991 Sep
PMID:Pregnenolone sulfate: a positive allosteric modulator at the N-methyl-D-aspartate receptor. 165 10

We have introduced the novel application of a simple ethidium fluorescence assay, using covalently closed circular DNA, for the study of topoisomerase-targeted drugs. With the specificity of camptothecin for eukaryotic topoisomerases I and of VM26 for eukaryotic topoisomerases II, the two classes of enzymes can be assayed independently in crude extracts and during purification. These assays are fast, sensitive, and quantitative, have a large sample capacity, and eliminate the need for radioactive materials, filters, and agarose gels. We have demonstrated the use of this fluorescence assay to measure the inhibition of the relaxation and supercoiling activities of purified mammalian topoisomerases I and II and bacterial gyrase by nonintercalating drugs. Similarly, the production of drug-induced topoisomerase-mediated cleavable complexes was readily quantitated with both nonintercalating and intercalating drugs. When inhibition and cleavage with VM-26 were measured concurrently as a function of topoisomerase II concentration, a clear inverse relationship between topoisomerase II inhibition and cleavable complex production was observed. When the physiologically relevant salt K+L-glutamate- was used, quantitative relaxation by topoisomerase II was observed up to twice the salt concentration obtained with KCl. The enantiomer K+D-glutamate- gave exactly the same results, indicating that the enhancing role of glutamate- is non-stereospecific.
Mol Pharmacol 1991 Oct
PMID:Fluorometric assays for DNA topoisomerases and topoisomerase-targeted drugs: quantitation of catalytic activity and DNA cleavage. 165 89

Phencyclidine (PCP) receptors have been solubilized from rat forebrain membranes with the zwitterionic detergent 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate. Specific binding of the potent PCP receptor ligands [3H]thienyl-phencyclidine (TCP) and [3H]MK-801 was restored by incorporating extracted membrane protein into lipid vesicles prepared from a total brain lipid extract. A nearly quantitative recovery of solubilized receptor activity was achieved; this was dependent upon both the concentration of detergent used during membrane solubilization and the concentration of added lipid used during the reconstitution. The single, saturable, binding site measured for both [3H]TCP and [3H]MK-801 in solubilized and reconstituted preparations exhibited properties similar to those of the high affinity PCP binding site labeled by these ligands in brain membranes. The ability of ligands selective for this site (MK-801, TCP, and dexoxadrol) to competitively displace specific [3H]TCP binding was retained after solubilization and reconstitution, although IC50 values measured for these ligands were shifted to higher concentrations. Levoxadrol and haloperidol were ineffective at displacing the radioligand binding in both membrane and vesicle preparations. The additive and dose-dependent ability of glutamate and glycine to enhance [3H]TCP binding to the solubilized/reconstituted receptor further suggests that a direct interaction with the N-methyl-D-aspartate receptor/ion channel complex has been preserved in the vesicle preparations. The photoaffinity labeling of two polypeptides (Mr 98,000 and 59,000) by azido-[3H]PCP was demonstrated in the vesicle preparations; this was largely prevented by competitive displacement of the radioligand with PCP before photolysis. These results establish both an essential lipid dependency and polypeptide composition for the high affinity, haloperidol-insensitive, PCP receptor in brain.
Mol Pharmacol 1991 Nov
PMID:High efficiency reconstitution of a phencyclidine/MK-801 receptor binding site solubilized from rat forebrain membranes. 165 3

1. Gamma-aminobutryic acid (GABA), a major inhibitory transmitter of the vertebrate retina, is synthesized from glutamate by L-glutamate decarboxylase (GAD) and mediates neuronal inhibition at GABAA receptors. GAD consists of two distinct molecular forms, GAD65 and GAD67, which have similar distribution patterns in the nervous system (Feldblum et al., 1990; Erlander and Tobin, 1991). GABAA receptors are composed of several distinct polypeptide subunits, of which the GABAA alpha 1 variant has a particularly extensive and widespread distribution in the nervous system. The aim of this study was to determine the cellular localization patterns of GAD and GABAA alpha 1 receptor mRNAs to define GABA- and GABAA receptor-synthesizing neurons in the rat retina. 2. GAD and GABAA alpha 1 mRNAs were localized in retinal neurons by in situ hybridization histochemistry with 35S-labeled antisense RNA probes complementary to GAD67 and GABAA alpha 1 mRNAs. 3. The majority of neurons expressing GAD67 mRNA is located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL). Occasional GAD67 mRNA-containing neurons are present in the inner plexiform layer. Labeled neurons are not found in the distal INL or in the outer nuclear layer (ONL). 4. GABAA alpha 1 mRNA is expressed by neurons distributed to all regions of the INL. Some discretely labeled cells are present in the GCL. Labeled cells are not observed in the ONL. 5. The distribution of GAD67 mRNA demonstrates that numerous amacrine cells (conventional, interstitial, and displaced) and perhaps interplexiform cells synthesize GABA. These cells are likely to employ GABA as a neurotransmitter. 6. The distribution of GABAA alpha 1 mRNA indicates that bipolar, amacrine, and perhaps ganglion cells express GABAA receptors having an alpha 1 polypeptide subunit, suggesting that GABA acts directly upon these cells.
Cell Mol Neurobiol 1991 Oct
PMID:Cellular distribution of L-glutamate decarboxylase (GAD) and gamma-aminobutyric acidA (GABAA) receptor mRNAs in the retina. 166 Mar 50


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