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J F Penrose

Publications and source records attributed to J F Penrose.

26 records · Page 2Linked to original sources
PubMed1994

Expression cloning of a cDNA for human leukotriene C4 synthase, an integral membrane protein conjugating reduced glutathione to leukotriene A4.

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.

5-Lipoxygenase-Activating Proteins↗
PubMed1992

Purification of human leukotriene C4 synthase.

Leukotriene (LT) C4 synthase, the enzyme that catalyzes the conjugation of LTA4 with reduced glutathione to form LTC4, was purified to homogeneity from the KG-1 myeloid cell line after solubilization of the microsomes utilizing a combination of 0.4% sodium deoxycholate and 0.4% Triton X-102. The solubilized enzyme was then applied to an S-hexyl-glutathione-agarose column that was eluted by the use of 7.5 mM probenecid. After removal of the probenecid by sequential concentration and dilution in an Amicon concentrator, the enzyme was additionally purified and concentrated by binding to and elution from approximately 75 mg of S-hexyl-glutathione-agarose. The enzyme was further resolved by electrophoresis with a nondenaturing Tris-glycine gel, and the LTC4 synthase activity was localized to slices 3 and 4. When the remainder of the eluate from the nondenaturing gel was precipitated by acetone and analyzed by 14% SDS/PAGE with silver staining, a single protein band of 18 kDa was associated with LTC4 synthase activity and was not present in the eluates of slices lacking activity. The overall recovery was 12.5%. In a separate preliminary purification, in which the yield was only approximately 1%, the eluates of the nondenaturing gel had also revealed a single protein of 18 kDa by SDS/PAGE, which was present only in the eluates with LTC4 synthase activity. These data identify LTC4 synthase as a protein of 18 kDa, a size consistent with its membership in the microsomal glutathione S-transferase family.

Chromatography, Affinity↗
PubMedDate not supplied

LTC4 synthase. Enzymology, biochemistry, and molecular characterization.

LTC4S conjugates reduce glutathione to LTA4 and is positioned as the pivotal and only committed enzyme involved in the formation of cysteinyl LTs. Despite its function as an enzyme that conjugates glutathione to LTA4, it is abundantly clear that LTC4S differs from the classic glutathione S-transferase (GST) families. This distinction is based on narrow substrate specificity, inability to conjugate GSH to xenobiotics, differential susceptibility to inhibitors, lack of homology, and failure to be immunorecognized by specific microsomal GST antibodies. The presence of LTC4S protein is restricted to a limited number of hematopoietic cells to include mast cells, eosinophils, basophils, monocytes/macrophages, and platelets, with the platelet being unique in its lack of the complete biosynthetic pathway for cysteinyl LTs. The purification of the protein and the cloning of the cDNA have demonstrated that the kinetic parameters of LTC4S are similar for the isolated natural or recombinant proteins. The protein is an 18-kDa integral perinuclear membrane enzyme, which is functional as a homodimer. The cDNA encodes a 150 amino-acid polypeptide monomer with three hydrophobic domains interspersed by two hydrophilic loops. Homology and secondary structural predictions have revealed that LTC4S is a member of a novel gene family that includes FLAP, mGST II, and mGST III. Each of these molecules is an integral membrane protein with the capacity to participate in LT biosynthesis: LTC4S as the terminal and only committed enzyme in cysteinyl LT formation, FLAP as an arachidonic acid presentation protein, and mGST II and mGST III as unique dual-function enzymes with primary detoxification functions. Site directed mutagenic studies of LTC4S have revealed that two residues, R51 and Y93, are involved in the acid and base catalysis, respectively, of LTA4 and GSH. Alignment of molecules with LTA4 conjugating ability demonstrates conservation of amino acid residues R51 and Y93, which appear necessary for this specific enzymatic function. The 2.5-Kb gene for human LTC4S contains five small exons and four introns, and the 5' UTR contains consensus sequences for AP-1 and AP-2 sites as well as an SP-1 site. The chromosomal localization of this gene is 5q35, distal to that of cytokine, growth factor, and receptor genes that have relevance to the development of allergic inflammation. Furthermore, there is genetic linkage of this region of human chromosome 5 to atopy and asthma, whereas no linkage exists for the chromosomal localization of the other family members, FLAP and mGST II, distinguishing LTC4S as a unique member of the novel gene family. LTC4S is profoundly overexpressed in the aspirin-induced asthmatic phenotype and correlates with overproduction of cysteinyl LTs and bronchial hyperreactivity to lysine aspirin. Ongoing studies are directed to the genomic regulation and additional polymorphisms within the gene of this pivotal enzyme, as well as to further identification of the amino acid residues central to its catalytic function.

Amino Acid Sequence↗
PubMedDate not supplied

The biochemical, molecular, and genomic aspects of leukotriene C4 synthase.

Leukotriene C4 (LTC4) synthase is an 18 kD integral membrane enzyme of the 5-lipoxygenase/LTC4 synthase pathway and is positioned as the pivotal and only committed enzyme for the formation of the cysteinyl leukotrienes. Although its function is to conjugate catalytically LTA4 to reduced glutathione, LTC4 synthase is differentiated from other glutathione S-transferase family members by its lack of amino acid homology, substrate specificity, and kinetics. LTC4 synthase (LTC4S) protein is present in the perinuclear membranes of a limited number of hematopoietic cells involved in allergic inflammation, including mast cells, eosinophils, basophils, and macrophages. The cDNA encodes a monomeric protein of 150 amino acids with three hydrophobic domains interspersed with two hydrophilic loops. Site-directed mutagenic studies reveal that the enzyme functions as a homodimer and that arginine-51 in the first hydrophilic loop, and tyrosine-93 in the second hydrophilic loop, are involved in the acid and base catalysis of LTA4 and glutathione, respectively. Homology and secondary structural predictions indicate that LTC4S is a novel member of a new gene superfamily of integral membrane proteins, each with the capacity to participate in leukotriene biosynthesis. The gene for LTC4S is 2.5 kb in length and is localized on chromosome 5q35, distal to that of the genes for cytokines and receptors important in the development and perpetuation of allergic inflammation. Immunohistochemical studies of mucosal biopsies from the bronchi of aspirin-intolerant asthmatics show that LTC4S is overrepresented in individuals with this phenotype, and this finding correlates with overproduction of cysteinyl leukotrienes and lysine-aspirin bronchial hyperreactivity.

Amino Acid Sequence↗
PubMedDate not supplied

Leukotriene C4 synthase: a candidate gene for the aspirin-intolerant asthmatic phenotype.

The features of the allergic inflammation underlying asthma occur as a consequence of mediators such as the cysteinyl LTs. The generation of the cysteinyl LTs is carefully regulated and dependent on the 5-LO/LTC4S pathway, of which LTC4S is the pivotal and only committed enzyme involved. Although LTC4S is related to other proteins involved in eicosanoid metabolism, it is clearly a distinct member within a novel gene family, and site directed mutagenic studies of LTC4S have identified two critical residues necessary for its specific conjugation of LTA4 to GSH. This observation, as well as the limited cellular distribution, and chromosomal localization are consistent with LTC4S as a candidate gene for asthma, having diverged from its other gene family members. More specifically, profound overexpression of LTC4S in aspirin-induced asthma seems to be a principal determinant of the respiratory reactions to aspirin, and a single-nucleotide polymorphism in the 5' regulatory region associates significantly with the aspirin-intolerant phenotype in Polish patients. This data strongly support LTC4S as a candidate gene in this phenotype of asthma, and further characterization of LTC4S in terms of enzymatic function and gene regulation will likely contribute to the understanding of the gene as one potentially responsible for the allergic inflammation underlying aspirin-intolerance. Additionally, discovery of additional polymorphism within this gene may lead to identification of susceptibility to adverse aspirin reactions, and inhibition of this enzyme may represent a therapeutic target for compounds useful in the treatment of asthma and allergic diseases.

Aspirin↗