Vesicular Gland

14,000,000 Leading Edge Experts on the ideXlab platform

Scan Science and Technology

Contact Leading Edge Experts & Companies

Scan Science and Technology

Contact Leading Edge Experts & Companies

The Experts below are selected from a list of 150 Experts worldwide ranked by ideXlab platform

William L Smith - One of the best experts on this subject based on the ideXlab platform.

  • photolabeling of prostaGlandin endoperoxide h synthase 1 with 3 trifluoro 3 m iiodophenyl diazirine as a probe of membrane association and the cyclooxygenase active site
    Journal of Biological Chemistry, 1996
    Co-Authors: James C Otto, William L Smith
    Abstract:

    Abstract Previous studies of the crystal structure of the ovine prostaGlandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M.(1994) Nature 367, 243-249) suggest that the enzyme is associated with membranes through a series of four amphipathic helices located between residues 70 and 117. We have used the photoactivatable, hydrophobic reagent 3-trifluoro-3-(m-[I]iodophenyl)diazirine ([I]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 are subject to photolabeling. After incubation of ovine Vesicular Gland microsomes with [I]TID, ovine PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg with trypsin established that [I]TID was incorporated into both the 33-kDa tryptic peptide containing the amino terminus and the 38-kDa tryptic peptide containing the carboxyl terminus. This pattern of photolabeling was not affected by the presence of 20 mM glutathione, indicating that the photolabeling observed for PGHS-1 was not due to the presence of [I]TID in the aqueous phase. However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [I]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa fragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide containing residues 25-166 which was labeled with [I]TID. This peptide contains the putative membrane binding domain of ovine PGHS-1. Our results provide biochemical support for the concept developed from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH terminus of the protein.

  • photolabeling of prostaGlandin endoperoxide h synthase 1 with 3 trifluoro 3 m 125i iodophenyl diazirine as a probe of membrane association and the cyclooxygenase active site
    Journal of Biological Chemistry, 1996
    Co-Authors: James C Otto, William L Smith
    Abstract:

    Abstract Previous studies of the crystal structure of the ovine prostaGlandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M.(1994) Nature 367, 243-249) suggest that the enzyme is associated with membranes through a series of four amphipathic helices located between residues 70 and 117. We have used the photoactivatable, hydrophobic reagent 3-trifluoro-3-(m-[I]iodophenyl)diazirine ([I]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 are subject to photolabeling. After incubation of ovine Vesicular Gland microsomes with [I]TID, ovine PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg with trypsin established that [I]TID was incorporated into both the 33-kDa tryptic peptide containing the amino terminus and the 38-kDa tryptic peptide containing the carboxyl terminus. This pattern of photolabeling was not affected by the presence of 20 mM glutathione, indicating that the photolabeling observed for PGHS-1 was not due to the presence of [I]TID in the aqueous phase. However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [I]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa fragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide containing residues 25-166 which was labeled with [I]TID. This peptide contains the putative membrane binding domain of ovine PGHS-1. Our results provide biochemical support for the concept developed from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH terminus of the protein.

Mats Hamberg - One of the best experts on this subject based on the ideXlab platform.

  • stereochemistry of oxygenation of linoleic acid catalyzed by prostaGlandin endoperoxide h synthase 2
    Archives of Biochemistry and Biophysics, 1998
    Co-Authors: Mats Hamberg
    Abstract:

    Linoleic acid was incubated with prostaGlandin–endoperoxide H synthase-2 (PGHS-2) from ovine placenta. A product consisting of regio- and stereoisomeric hydroxyoctadecadienoic (HOD) acids was obtained. Analysis by straight-phase high-performance liquid chromatography followed by chiral-phase high-performance liquid chromatography demonstrated that linoleic acid was preferentially oxygenated at C-9 to produce the following mixture of HODs: 9(R)-HOD (52%), 9(S)-HOD (11%), 13(R)-HOD (2%), and 13(S)-HOD (35%). As a comparison, linoleic acid was incubated with microsomal prostaGlandin–endoperoxide H synthase-1 (PGHS-1) from ovine Vesicular Gland. This resulted in a product having the following composition: 9(R)-HOD (73%), 9(S)-HOD (9%), 13(R)-HOD (1%), and 13(S)-HOD (17%). The stereochemistry of the hydrogen which was removed from C-11 during the conversion of linoleic acid into hydroxy acids in the presence of PGHS-1 or PGHS-2 was determined by incubation of [(11R)-2H]- and [(11S)-2H]linoleic acids followed by mass spectrometric analysis of the isotope contents of the individual hydroxy acid isomers. Both enzymes were found to catalyze oxygenations which involved stereospecific removal of the (11S) hydrogen and retention of the (11R) hydrogen. The major hydroxy acids, i.e., 9(R)-HOD and 13(S)-HOD, were formed from linoleic acid in reactions which involved antarafacial hydrogen abstraction and oxygen insertion. It is concluded that the initial steps of the PGHS-2- and PGHS-1-catalyzed oxygenations proceed with identical stereochemistry and involve stereospecific removal of the pro-Shydrogen from the ω8-methylene group of the substrate.

  • oxygenation of 5 8 11 eicosatrienoic acid by prostaGlandin endoperoxide synthase and by cytochrome p450 monooxygenase structure and mechanism of formation of major metabolites
    Archives of Biochemistry and Biophysics, 1993
    Co-Authors: Ernst H. Oliw, Howard Sprecher, Lena Hörnsten, Mats Hamberg
    Abstract:

    Abstract Incubation of 5,8,11-[1- 14 C]eicosatrienoic acid with prostaGlandin endoperoxide synthase of ram Vesicular Gland microsomes led to formation of a number of polar metabolites. Four major compounds were characterized by chemical and physical methods and found to be: (13 R )-hydroxy-5,8,11-eicosatrienoic acid, (11 R )-hydroxy-5,8,12-eicosatrienoic acid, 8,9,11-trihydroxy-5,12-eicosadienoic acid (two diastereoisomers), and 8,9-epoxy-11-hydroxy-5,12-eicosadienoic acid. On the basis of previous studies on the mechanism of prostaGlandin biosynthesis it seemed likely that the initial step of conversion of 5,8,11-eicosatrienoic acid consisted of removal of the pro-S hydrogen from C-13. The resulting carbon-centered radical was apparently attacked by dioxygen at C-13 to provide a (13 R )-(hydro)peroxy derivative, which served as the precursor of (13 R )-hydroxyeicosatrienoic acid. Alternatively, attack by dioxygen occurred at C-11 to produce an (11 R )-peroxy radical. This intermediate was further converted to (11 R )-hydroxyeicosatrienoic acid by reduction, into two 8,9,11-trihydroxy-5,12-eicosadienoic acids by successive cyclization, oxygenation, and reduction, and into the epoxy-hydroxy acid by cyclization and intramolecular epoxidation. The relative abundance of (13 R )-hydroxy-5,8,11-eicosatrienoic acid, (11 R )-hydroxy-5,8,12-eicosatrienoic acid, and the epoxy alcohol plus the two 8,9,11-triols was 51, 9, and 40%, respectively. The oxygenation at C-13 and C-11 of 5,8,11-eicosatrienoic acid was inhibited by 90% in the presence of diclofenac, an inhibitor of prostaGlandin endoperoxide synthase. The two diastereomeric 8,9,11-trihydroxy acids and the epoxy-hydroxy acid are novel oxylipins and their formation provides independent chemical evidence for the existence of an 11-peroxy radical intermediate in prostaGlandin endoperoxide synthase catalysis. Oxygenation of 5,8,11-eicosatrienoic acid by cytochrome P450 from liver microsomes of cynomolgus monkeys and phenobarbital-treated rats was also investigated. The metabolites formed included 19- and 20-hy-droxyeicosatrienoic acid, 8,9- and 11,12-dihydroxyeicosadienoic acids (formed by enzymatic hydrolysis of the corresponding epoxides), and (12 R )-hydroxy-5,8,10-hydroxyeicosatrienoic acid.

James C Otto - One of the best experts on this subject based on the ideXlab platform.

  • photolabeling of prostaGlandin endoperoxide h synthase 1 with 3 trifluoro 3 m iiodophenyl diazirine as a probe of membrane association and the cyclooxygenase active site
    Journal of Biological Chemistry, 1996
    Co-Authors: James C Otto, William L Smith
    Abstract:

    Abstract Previous studies of the crystal structure of the ovine prostaGlandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M.(1994) Nature 367, 243-249) suggest that the enzyme is associated with membranes through a series of four amphipathic helices located between residues 70 and 117. We have used the photoactivatable, hydrophobic reagent 3-trifluoro-3-(m-[I]iodophenyl)diazirine ([I]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 are subject to photolabeling. After incubation of ovine Vesicular Gland microsomes with [I]TID, ovine PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg with trypsin established that [I]TID was incorporated into both the 33-kDa tryptic peptide containing the amino terminus and the 38-kDa tryptic peptide containing the carboxyl terminus. This pattern of photolabeling was not affected by the presence of 20 mM glutathione, indicating that the photolabeling observed for PGHS-1 was not due to the presence of [I]TID in the aqueous phase. However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [I]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa fragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide containing residues 25-166 which was labeled with [I]TID. This peptide contains the putative membrane binding domain of ovine PGHS-1. Our results provide biochemical support for the concept developed from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH terminus of the protein.

  • photolabeling of prostaGlandin endoperoxide h synthase 1 with 3 trifluoro 3 m 125i iodophenyl diazirine as a probe of membrane association and the cyclooxygenase active site
    Journal of Biological Chemistry, 1996
    Co-Authors: James C Otto, William L Smith
    Abstract:

    Abstract Previous studies of the crystal structure of the ovine prostaGlandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M.(1994) Nature 367, 243-249) suggest that the enzyme is associated with membranes through a series of four amphipathic helices located between residues 70 and 117. We have used the photoactivatable, hydrophobic reagent 3-trifluoro-3-(m-[I]iodophenyl)diazirine ([I]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 are subject to photolabeling. After incubation of ovine Vesicular Gland microsomes with [I]TID, ovine PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg with trypsin established that [I]TID was incorporated into both the 33-kDa tryptic peptide containing the amino terminus and the 38-kDa tryptic peptide containing the carboxyl terminus. This pattern of photolabeling was not affected by the presence of 20 mM glutathione, indicating that the photolabeling observed for PGHS-1 was not due to the presence of [I]TID in the aqueous phase. However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [I]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa fragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide containing residues 25-166 which was labeled with [I]TID. This peptide contains the putative membrane binding domain of ovine PGHS-1. Our results provide biochemical support for the concept developed from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH terminus of the protein.

Bengt Samuelsson - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of Microsomal, Glutathione Dependent ProstaGlandin E Synthase
    Advances in experimental medicine and biology, 2002
    Co-Authors: Per-johan Jakobsson, Ralf Morgenstern, Staffan Thorén, Bengt Samuelsson, Bengt Samuelsson
    Abstract:

    Terminal prostanoid synthases constitute a group of several specific enzymes that catalyze the further metabolism of cyclooxygenase-derived prostaGlandin H2. The efficient biosynthesis of prostaGlandin E2 requires prostaGlandin E synthase (E.C. 5.3.99.3). High prostaGlandin E synthase (PGES) activity is found in the sheep and bovine Vesicular Gland. In this organ, both PGES and cyclooxygenase activities are localized to the microsomal membrane system, where the PGES activity is dependent on milli-molar concentrations of reduced glutathione. The activity of the enzyme rapidly deteriorated following solubilization, why any attempts to identify this protein by purification have not been successful1,2. A different method employed to try identifying the sheep microsomal PGES involved immunoprecipitation3. Two membrane associated PGES’s with molecular masses of 17.5 kDa and 180 kDa were reported. The smaller enzyme possessed a lower Km (40 1M) for PGH2than the larger protein (150.tM) and interestingly, the smaller enzyme was also localized to the same membrane system as cyclooxygenase3. None of these enzymes possessed any significant glutathione-S- transferase (GST) activity. In contrast, purification of the enzymes responsible for cytosolic PGES activity in human brain as well asAscaridia gallihave revealed their nature as cytosolic GST’s4,5. In addition to the glutathione dependent PGES’s, a glutathione independent, microsomal PGES has been characterized6which was recently purified from bovine heart7.

Garret A. Fitzgerald - One of the best experts on this subject based on the ideXlab platform.

  • human platelet erythroleukemia cell prostaGlandin g h synthase cdna cloning expression and gene chromosomal assignment
    The FASEB Journal, 1991
    Co-Authors: Colin D Funk, Lena B Funk, Matthew E Kennedy, Amy S Pong, Garret A. Fitzgerald
    Abstract:

    Platelets metabolize arachidonic acid to thromboxane A2, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaGlandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaGlandin endoperoxide-derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep Vesicular Gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in th...