Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C1260386 (GSH)
 38,102 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Leukotriene (LT) A4 metabolism was studied in human platelets and endothelial cells, since both cells could be involved in transcellular formation of LTC4. Upon addition of exogenous LTA4, both cells produced LTC4 as a major metabolite at various incubation times, and no LTB4, LTD4, or LTE4 was detected. Kinetic studies revealed a higher apparent Km for LTA4 in endothelial cells as compared to platelets (5.8 microM for human umbilical vein endothelial cells (HUVEC) versus 1.3 microM for platelets); platelets were more efficient in this reaction with a higher Vmax (174 pmol/mg protein/min) versus 15 pmol/mg protein/min in HUVEC. The formation of LTC4 and corresponding kinetic parameters were not modified when platelets or endothelial cells were stimulated by thrombin prior to or simultaneously with the addition of LTA4. In both cells LTC4 synthase activity was not modified by repeated addition of LTA4 showing that it is not a suicide-inactivated enzyme. Furthermore, in platelets and endothelial cells, the enzyme activity was localized in the membrane fraction and was distinct from cytosolic glutathione-S-transferases. Platelet membrane fractions showed apparent Km values of 31 microM and 1.2 mM for LTA4 and GSH, respectively. Inhibition of LTC4 formation from platelets and endothelial cells preparations by S-substituted glutathione derivatives was correlated to the length of the S-alkyl chain. The same substances inhibited cytosolic glutathione-S-transferases with significantly lower IC50, confirming the distinct nature of the two enzymes. These results show that platelets and HUVEC possess similar enzymes for the production of LTC4 from LTA4; however, platelets seem to have a higher efficiency than HUVEC in performing this reaction.
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PMID:Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells. 132 61

The utilization of LTA4 by peritoneal macrophages (MO) obtained from untreated rats (control) as well as by those elicited from rats was investigated at designated intervals (on days 3, 7, and 14) following the intraperitoneal injection of thioglycollate (TG). On day 7 following the injection the elicited MO converted LTA4 to LTC4 at the highest rate while the resident MO showed the lowest rate. The conversion of LTA4 to LTC4 and LTB4 was next examined by using each MO lysate. The apparent LTC4 synthase activity was significantly higher in the MO lysate both on day 3 and day 7, with the latter being the highest value obtained. The GSH S-transferase activity in each lysate using as the substrate, DNCB was significantly lower on day 3 but significantly higher on day 7 as compared to control values. However, this elevated activity was less variable than that observed with LTC4 synthase. The possible implication for these observations is discussed.
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PMID:Enhanced leukotriene C4 synthase activity in thioglycollate-elicited peritoneal macrophages. 222 48

Leukotriene C4 (LTC4) synthase, which conjugates LTA4 and LTA4-methyl ester (LTA4-me) with glutathione (GSH) to form LTC4 and LTC4-me, respectively, has been solubilized from the microsomes of guinea pig lung and purified 91-fold in four steps to a specific activity of 692 nmol/10 min per mg protein using LTA4-me as substrate. LTC4 synthase of guinea pig lung was separated from microsomal GSH S-transferase by Sepharose CL-4B chromatography and further purified by DEAE-Sephacel chromatography, agarose-butylamine chromatography, and DEAE-3SW fast-protein liquid chromatography. It was also differentiated from the microsomal GSH S-transferase, which utilized 1-chloro-2,4-dinitrobenzene as a substrate, by its heat lability and relative resistance to inhibition by S-hexyl-GSH. The Km value of guinea pig lung LTC4 synthase for LTA4 was 3 microM and the Vmax was 108 nmol/3 min per microgram; the Km values for LTA3 and LTA5 were similar, and the Vmax values were about one-half those obtained with LTA4. The conversion of LTA4-me to LTC4-me was competitively inhibited by LTA3, LTA4, and LTA5, with respective Ki values of 1.5, 3.3, and 2.8 microM, suggesting that these substrates were recognized by a common active site. IC50 values for the inhibition of the conjugation of 20 microM LTA4-me with 5 mM GSH were 2.1 microM and 0.3 microM for LTC4 and LTC3, respectively. In contrast, LTD4 was substantially less inhibitory (IC50 greater than 40 microM), and LTE4 and LTB4 had no effect on the enzyme, indicating that the mixed type product inhibition observed was specific for sulfidopeptide leukotrienes bearing the GSH moiety.
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PMID:Properties of highly purified leukotriene C4 synthase of guinea pig lung. 334 45

Leukotriene (LT) C4 synthase catalyzes the conjugation of LTA4 with reduced glutathione (GSH) to form LTC4. This enzyme was purified to homogeneity from Kirsten Sarcoma transformed murine mast cells and from the human monocytic cell line THP-1 by two steps: a microsomal extract was partially purified by affinity chromatography on S-hexyl GSH agarose. LTC4 synthase was separated from other GSH-binding proteins by preparative gel electrophoresis under non denaturing conditions. The proteins obtained from human and murine cells showed a single band on a SDS gel with a molecular mass of 14 to 17 kDa, depending on the gel system. N-terminal sequencing revealed high homology between the LTC4 synthases of both species.
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PMID:Two step purification of human and murine leukotriene C4 synthase. 776 6

Cysteinyl leukotrienes (LT) play an important role in the development of experimental glomerulonephritis (GN). We have partially purified and characterized LTC4 synthase, the enzyme responsible for cysteinyl LT formation, from rat renal microsomes and have investigated this enzyme activity in nephritic rats. LTC4 formation, measured in vitro, was linear for > 10 min at 25 degrees C in the presence of 50 mM serine borate (an inhibitor of gamma-glutamyl transpeptidase), with Km values for LTA4 and GSH of 56 microM and 8.5 mM, respectively. Detergent solubilization and anion-exchange chromatography of microsomal proteins resulted in a 7-fold increase in enzyme specific activity. Enzymatic and immunoblot analysis demonstrated that cytosolic and microsomal glutathione S-transferase (GST) activities were distinct from LTC4 synthase activity. Comparison of LTC4 synthase activity in nephritic rats over 21 days revealed an initial increase over the first 24 h following injection of nephrotoxic sera, followed by a subsequent decline until day 7 and a gradual recovery by day 21. Inhibition of LT biosynthesis with MK-0591 (10 mg kg-1 d-1) reduced GN-associated proteinuria by 72% (P < 0.05). These results suggest a potential mechanism for enhanced cysteinyl LT formation in the development of experimental GN and further support their causal role in the etiology of this disease.
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PMID:Renal leukotriene C4 synthase: characterization, partial purification and alterations in experimental glomerulonephritis. 782 26

Leukotriene (LT) C4 synthase, an integral microsomal membrane protein, conjugates LTA4, an epoxide intermediate, to reduced glutathione (GSH) to form a proinflammatory mediator, LTC4. A sensitive fluorescence-linked immunoassay for LTC4 was used to screen a KG-1 cDNA expression library for LTC4 synthase activity after transfection of COS cells and addition of substrate LTA4. Stepwise resolution of 240,000 colonies in 96 pools led to the identification of individual clones with maximal LTC4 synthase activity that contained a 694-bp cDNA insert. This insert was composed of a 54-bp 5' nontranslated region, an ATTAAA polyadenylylation signal, and a poly(A)+ tail. The open reading frame encodes a 16.5-kDa protein with a pI of 11.05. Hybridization with a cDNA probe demonstrated a mRNA transcript of 0.7 kbp in RNAs from human eosinophils and KG-1 cells, which contain LTC4 synthase. The nucleotide and deduced amino acid sequences of the LTC4 synthase cDNA show no significant homology to GSH S-transferases but share 31% overall amino acid identity with 5-lipoxygenase activating protein (FLAP). The identity at the N-terminal two-thirds of these two proteins is 44%, with some regions of near identity. Peptide structural analysis of the deduced LTC4 synthase predicts the presence of three transmembrane domains nearly superimposable on those of FLAP. Moreover, LTC4 synthase is inhibitable by a FLAP inhibitor, MK-886. Therefore, LTC4 synthase is distinct from the known GSH S-transferases by nucleotide and consensus amino acid sequences, and its GSH-conjugating function represents a distinct integral membrane protein belonging to a distinct gene family.
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PMID:Expression cloning of a cDNA for human leukotriene C4 synthase, an integral membrane protein conjugating reduced glutathione to leukotriene A4. 805 39

Overnight (10-16 h) incubation of retinoic acid (RA), a derivative of vitamin A, specifically induced LTC4 synthase activity (5 to 10-fold), but not LTA4 hydrolase activity in the lysate of rat basophilic leukemia-1 (RBL-1) cells. A time course study revealed that the increase of LTC4 synthase activity was time dependent and that the peak value was obtained after a 24-hour incubation with RA. The induction of enzyme activity was specifically localized to the microsomal fraction. Glutathione (GSH) S-transferase activity measured by using the same cell lysate as an enzyme source and 1-chloro-2,4-dinitrobenzene (DNCB) as a substrate was not influenced by RA treatment, indicating that the induction by RA is specific for membrane-bound LTC4 synthase. The induction of LTC4 synthase may be an important regulatory mechanism of peptide-LT synthesis in allergy and inflammatory diseases.
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PMID:Specific induction of LTC4 synthase by retinoic acid in rat basophilic leukemia-1 cells. 811 Dec 44

Leukotriene C4 (LTC4) synthase catalyzes the conjugation of LTA4 with reduced GSH to form LTC4, the parent of the receptor active cysteinyl leukotrienes implicated in the pathobiology of bronchial asthma. Previous cloning of the cDNA for human LTC4 synthase demonstrated significant homology of its amino acid sequence to that of 5-lipoxygenase activating protein (FLAP) but none to that of the GSH S-transferase super-family. Genomic cloning from a P1 library now reveals that the gene for LTC4 synthase contains five exons (ranging from 71 to 257 nucleotides in length) and four introns, which in total span 2.52 kilobase pairs in length. The intron/exon junctions of LTC4 synthase align identically with those of FLAP; however, the small size of the LTC4 synthase gene contrasts with the > 31-kilobase pair size reported for FLAP. Confirmation of the LTC4 synthase gene size to ensure that no deletions had occurred during the cloning was obtained by two overlapping polymerase chain reactions from genomic DNA, which provided products of the predicted sizes. Primer extension analysis with poly(A)+ RNA from culture-derived human eosinophilic granulocytes or the KG-1 myelogenous cell line revealed multiple transcriptional start sites with prominent signals at 66, 69, and 96 base pairs 5' of the ATG translation start site. The 5'-flanking region revealed a GC-rich promoter sequence consistent with an SP-1 site and consensus sequences for AP-1 and AP-2 enhancer elements, 24, 807, and 877 bp, respectively, 5' from the first transcription initiation site. Southern blot analysis of a genomic DNA (with full-length cDNA as well as 5' and 3' oligonucleotide probes) confirmed the size of the gene and indicated a single copy gene in normal human genomic DNA. Fluorescent in situ hybridization mapped LTC4 synthase to chromosomal location 5q35, which is in close proximity to the cluster of genes for cytokines and receptors involved in the regulation of cells central to allergic inflammation and implicated in bronchial asthma.
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PMID:Molecular cloning of the gene for human leukotriene C4 synthase. Organization, nucleotide sequence, and chromosomal localization to 5q35. 862 89

Leukotriene C4 synthase (EC 2.5.1.37) catalyzes the conjugation of reduced glutathione (GSH) with leukotriene A4 to form the intracellular parent of the proinflammatory cysteinyl leukotrienes. Human leukotriene C4 synthase shares substantial amino acid identity in its consensus N-terminal two-thirds with 5-lipoxygenase-activating protein and has a region (residues 37-58) that exhibits 46% amino acid identity with a domain of this protein (residues 41 -62) to which an inhibitor binds. We have now cloned mouse leukotriene C4 synthase CDNA using the polymerase chain reaction to screen a mouse pcDNA3 expression library with oligonucleotide primers based on the translated human leukotriene C4 synthase cDNA sequence. Mouse leukotriene C4 synthase cDNA is 667 bp in length, including the poly(A)-rich tail, and shows 87% similarity with the human cDNA within the open reading frame. The deduced 150-amino-acid sequence of mouse leukotriene C4 synthase (differs from the human enzyme by only 18 amino acids, of which 9 reside at the C terminus. The potential N-glycosylation site, two protein kinase C phosphorylation sites, the two cysteine residues, and the putative inhibitor-binding domain (substitutions Thr4l-->Ser and Tyr50-->Phe) were conserved in mouse leukotriene C4 synthase. Northern blot analysis indicated that the leukotriene C4 synthase RNA transcript is widely distributed. The Km values for leukotriene A4 methyl ester, leukotriene A4 free acid and GSH were 7.6 microM, 3.6 microM and 1.6 mM, respectively, for purified human recombinant enzyme, and 10.3 microM, 2.5 microM and 1.9 microM, respectively, for purified recombinant mouse enzyme; the corresponding Vmax values were 2.5, 1.3 and 2.7 micromol x min(-1) x mg(-1) protein, respectively, for human enzyme, and 2.3, 1.2 and 2.2 micromol x min(-1) x mg(-1) protein, respectively, for mouse enzyme. The 5-lipoxygenase-activating-protein inhibitor, MK-886, was active against both human and mouse recombinant leukotriene C4 synthase with IC50 values of 3.1 microM and 2.7 microM respectively. These findings confirm that the leukotriene C4 synthases belong to a gene family that includes the 5-lypoxygenase-activating protein and suggest that the C-terminal domain of leukotriene C4 synthase may not be critical for its conjugation function.
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PMID:Molecular cloning, expression and characterization of mouse leukotriene C4 synthase. 870 58

The functional characteristics of leukotriene C4 synthase (LTC4S), which specifically conjugates leukotriene A4 with GSH, were assessed by mutagenic analysis. Human LTC4S and the 5-lipoxygenase-activating protein share substantial amino acid identity and predicted secondary structure. The mutation of Arg-51 of LTC4S to Thr or Ile abolishes the enzyme function, whereas the mutation of Arg-51 to His or Lys provides a fully active recombinant protein. The mutations Y59F, Y97F, Y93F, N55A, V49F, and A52S increase the Km of the recombinant microsomal enzyme for GSH. The mutation Y93F also markedly reduces enzyme function and increases the optimum for pH-dependent activity. The deletion of the third hydrophobic domain with the carboxyl terminus abolishes the enzyme activity, and function is restored by the substitution of the third hydrophobic domain and carboxyl terminus of 5-lipoxygenase-activating protein for that of LTC4S. Mutations of C56S and C82V alone or together and the deletion of Lys-2 and Asp-3 of LTC4S do not alter enzyme function. The direct linkage of two LTC4S monomers by a 12-amino acid bridge provides an active dimer, and the same bridging of inactive R51I with a wild-type monomer creates an active pseudo-dimer with function similar to that of the wild-type enzyme. These results suggest that in the catalytic function of LTC4S, Arg-51 probably opens the epoxide ring and Tyr-93 provides the thiolate anion of GSH. Furthermore, the monomer has independent conjugation activity, and dimerization of LTC4S maintains the proper protein structure.
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PMID:Site-directed mutagenesis of human leukotriene C4 synthase. 915 54


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