Publications

• Synthesis and cannabinoid receptor binding activity of conjugated triene anandamide, a novel eicosanoid.

  Wise M.L., Soderstrom K., Murray T.F., Gerwick W.H.

  A polyenoic fatty-acid isomerase (PFI) from a red marine alga was used to convert anandamide (5Z,8Z,11Z,14Z-eicosatetraenoyl-N-ethan olamide) to the 5Z,7E,9E,14Z-eicosatetraenoyl-N-ethanol amide isomer. This novel eicosanoid, termed conjugated triene anandamide (CTA), was assessed for its ability ... >> More

  A polyenoic fatty-acid isomerase (PFI) from a red marine alga was used to convert anandamide (5Z,8Z,11Z,14Z-eicosatetraenoyl-N-ethan olamide) to the 5Z,7E,9E,14Z-eicosatetraenoyl-N-ethanol amide isomer. This novel eicosanoid, termed conjugated triene anandamide (CTA), was assessed for its ability to bind to the cannabinoid receptor in rat brain membrane preparations. CTA is a high affinity cannabimimetic substance whose novel structure provides new insight into structure-activity relationships of cannabinoid receptor ligands. These experiments illustrate the utility of enzymes isolated from marine organisms in the development of pharmacological probes. << Less

  Experientia 52:88-92(1996) [PubMed] [EuropePMC]

• Biosynthesis of conjugated triene-containing fatty acids by a novel isomerase from the red marine alga Ptilota filicina.

  Wise M.L., Hamberg M., Gerwick W.H.

  The biosynthesis of conjugated triene-containing fatty acids by the red alga Ptilota filicina is catalyzed by a novel enzyme, polyenoic fatty acid isomerase. The enzyme has been highly purified and is described here for the first time. Matrix-assisted laser-induced desorption mass spectrometry was ... >> More

  The biosynthesis of conjugated triene-containing fatty acids by the red alga Ptilota filicina is catalyzed by a novel enzyme, polyenoic fatty acid isomerase. The enzyme has been highly purified and is described here for the first time. Matrix-assisted laser-induced desorption mass spectrometry was used to determine that the major protein in the purified enzyme is composed of similar or identical subunits of M(r) 58,119 Da. The native enzyme emerges with an apparent M(r) of 174,000 Da from a gel permeation chromatography column. While this enzyme catalyzes the formation of conjugated trienes from a variety of polyunsaturated fatty acid precursors [arachidonate ((5Z,8Z,11Z,14Z)-eicosatetraenoate) is converted to (5Z,7E,9E,14Z)-eicosatetraenoate; gamma-linolenate ((6Z,9Z,12Z)-octadecatrienoate) is converted to 6Z,8E,-10E-octadecatrienoate], this occurs most rapidly with eicosapentaenoate [(5Z,7E,9E,14Z,17Z)-eicosapentaenoate], which is likely the native substrate. Through a series of experiments utilizing gamma-linolenates stereospecifically labeled with deuterium, we have determined that the enzyme intramolecularly transfers the bis-allylic pro-S hydrogen from the C11 position to the C13 position. Furthermore, the bis-allylic pro-R hydrogen at C8 in gamma-linolenate is lost to the solvent. Using arachidonate as substrate, we demonstrated that the C11 olefinic position becomes protonated by a solvent-derived proton. There appears to be no requirement for molecular oxygen, and the transformation is catalyzed by this single enzyme. << Less

  Biochemistry 33:15223-15232(1994) [PubMed] [EuropePMC]

• Characterization of the substrate binding site of polyenoic fatty acid isomerase, a novel enzyme from the marine alga Ptilota filicina.

  Wise M.L., Rossi J., Gerwick W.H.

  The substrate binding site of polyenoic fatty acid isomerase (PFI) has been investigated using a series of alternate substrates and by examination of the pH dependence on the kinetic parameters of PFI with selected substrates. The pH dependence profile of PFI with EPA [(5Z,8Z,11Z,14Z,17Z)-eicosape ... >> More

  The substrate binding site of polyenoic fatty acid isomerase (PFI) has been investigated using a series of alternate substrates and by examination of the pH dependence on the kinetic parameters of PFI with selected substrates. The pH dependence profile of PFI with EPA [(5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid] shows the enzyme to be catalytically active over a wide pH range, with activity being optimal below pH 6.0. Analysis of the kinetic parameters of DHA [(4Z,7Z,10Z,13Z,16Z,19Z)-docosahexen oic acid]; adrenic acid [(7Z,10Z,13Z,16Z)-docosatetraenoic acid]; EPA; arachidonic acid [(5Z,8Z,11Z,14Z)-eicosatetraenoic acid]; anandamide (arachidonyl-N-ethanolamide); and eicosatrienoic acid [(5Z,8Z,11Z)-eicosatrienoic acid] demonstrates that substrates possessing omega-3 olefins (DHA and EPA) have the lowest K(m) values (1.9 and 9.6 microM, respectively). EPA and arachidonic acid showed the highest V(max) values (6.0 and 2.8 micromol min(-1) mg(-1), respectively). The twenty carbon omega-9 fatty acid eicosatrienoic acid showed a relatively large K(m) and had a V(max) approximately 20-fold less than EPA. Anandamide, a substrate analog lacking an ionizable carboxylate, showed a K(m) similar to the other omega-6 fatty acids (arachidonic acid and adrenic acid); however, the V(max) was approximately 5-fold lower than arachidonic acid and 8-fold lower than EPA. Moreover, anandamide demonstrated no pH dependency on its kinetic parameters over a range where EPA showed a 27-fold decrease in V/K(m). NMR spectroscopy was used to determine the structure of the product from reaction of PFI with DHA. These data showed the compound to be (4Z,7Z,9E,11E,16Z,19Z)-docosahexenoi c acid. Reaction of PFI with dihomo-gamma-linolenic acid resulted in the development of two products, one with the characteristic chromophore of a conjugated triene, the other with a chromophore characteristic of a conjugated diene. Analysis of the products from these reactions of PFI, in conjunction with the kinetic parameters from the alternate substrates, provides compelling evidence that the enzyme preferentially orients the substrate in the catalytic site with respect to the methyl terminus. << Less

  Biochemistry 36:2985-2992(1997) [PubMed] [EuropePMC]

• Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: protein characterization and functional expression of the cloned cDNA.

  Zheng W., Wise M.L., Wyrick A., Metz J.G., Yuan L., Gerwick W.H.

  The recently described enzyme, polyenoic fatty acid isomerase (PFI), from the marine alga Ptilota filicina J. Argardh has been analyzed with respect to its protein structure and an associated cofactor. The enzyme was purified to homogeneity (as judged by SDS-PAGE and silver staining). By sedimenta ... >> More

  The recently described enzyme, polyenoic fatty acid isomerase (PFI), from the marine alga Ptilota filicina J. Argardh has been analyzed with respect to its protein structure and an associated cofactor. The enzyme was purified to homogeneity (as judged by SDS-PAGE and silver staining). By sedimentation equilibrium ultracentrifugation the mass of the native enzyme was estimated to be 125 kDa. The N-terminal peptide sequence derived from this protein was used to isolate two very similar cDNA clones encoding novel 500-amino acid proteins, both with calculated molecular masses of 55.9 kDa and pIs of 4.87. The data predict translation of a preprotein containing a signal peptide of 21 amino acids that is removed during maturation. Deglycosylation assays demonstrate that native PFI from P. filicina is a glycoprotein. The purified protein is chromophoric with a flavin-like UV spectrum and sequence analysis reveals the presence of a flavin-binding motif near the mature N-terminus. Heterologous expression of active PFI in Arabidopsis, using one of the cDNA clones, was successful as evidenced by conversion of arachidonic acid to a conjugated triene in an in vitro assay of the transgenic plant tissues. << Less

  Arch. Biochem. Biophys. 401:11-20(2002) [PubMed] [EuropePMC]