Expression of an 8R-Lipoxygenase From the Coral Plexaura homomalla.

Methods are presented for the use of the coral 8R-lipoxygenase from the Caribbean sea whip coral Plexaura homomalla as a model enzyme for structural studies of animal lipoxygenases. The 8R-lipoxygenase is remarkably stable and can be stored at 4°C for 3 months with virtually no loss of activity. In addition, an engineered "pseudo wild-type" enzyme is soluble in the absence of detergents, which helps facilitate the preparation of enzyme:substrate complexes.

Identification and characterization of an arachidonate 11R-lipoxygenase.

11R-Lipoxygenase (11R-LOX) activity has been detected in several marine invertebrates, and here we report the first cloning and expression of the enzyme. The cDNA encoding a protein of 77kDa was isolated by RT-PCR from the soft coral Gersemia fruticosa and expressed in Escherichia coli. Incubations of recombinant enzyme with arachidonic acid yielded a single product, identified by RP-HPLC, GC-MS, and chiral phase-HPLC as 11R-hydroperoxyeicosatetraenoic acid. Other C18, C20, and C22 substrates are also oxygenated, preferentially at the omega10 position. Significantly, both Ca(2+)-ions and a membrane fraction are required for catalytic activity. Calcium effects translocation of the soluble 11R-LOX to the membrane and this association is reversible by Ca(2+) chelation. The enzyme sequence contains some conserved amino acids implicated in calcium activation of mammalian 5-LOX, and with its obligate requirement for membrane interaction the 11R-LOX may thus provide a new model for further analysis of this aspect of lipoxygenase activation.

Isolation and characterization of 5-lipoxygenase from tulip bulbs.

An unique membrane bound lipoxygenase was isolated and purified from purple star tulip bulbs with a specific activity of 5.2 mu moles O2 consumed.min-1.mg-1 protein. The purified tulip enzyme exhibits regiospecificity for O2 insertion at C-5 of the arachidonic acid molecule. Identification of the reaction product was confirmed as 5-hydroperoxyeicosatetraenoic acid by analytical criteria which included: cochromatography with the authentic compound, as well as mass spectral and 1H-NMR analysis. Thus, the enzyme from tulip bulbs appears to be different from the cytosolic lipoxygenase from potato tubers, which exhibits non-regiospecificity in terms of O2 incorporation. However, the purified tulip lipoxygenase showed a strong immunological crossreactivity with antiserum raised against the purified potato lipoxygenase, indicating close immunological relationship with the other plant lipoxygenases.

Properties of the soluble arachidonic acid 15-lipoxygenase and 15-hydroperoxide isomerase from the oomycete Saprolegnia parasitica.

The soluble hydroperoxide isomerase and 15-lipoxygenase activities were partially purified from the oomycete Saprolegnia parasitica and some of their properties characterized. Both enzymes co-eluted with a molecular weight of 145,000-150,000 on Sephacryl S-300 chromatography. The enzyme activities also co-eluted on DEAE Sephadex ion exchange chromatography and hydroxylapatite chromatography. Both activities showed similar responses to pH and temperature. Both enzymes showed parallel inhibition by p-hydroxymercuribenzoate and eicosatetraynoic acid. The partially purified hydroperoxide isomerase showed an apparent km of 166 microM and a Vmax of 5.3 mumol/min/mg protein for exogenous 15-HPETE. It was not stimulated by calcium. These results suggest that the soluble hydroperoxide isomerase and 15-lipoxygenase activities from S. parasitica are both contained on the same protein or protein complex.

Purification, characterization, and structural properties of a single protein from rat basophilic leukemia (RBL-1) cells possessing 5-lipoxygenase and leukotriene A4 synthetase activities.

Arachidonate 5-lipoxygenase of rat basophilic leukemia (RBL-1) cells was purified more than 1000-fold by gel filtration and anion exchange protein-high performance liquid chromatography (HPLC). Physical properties of the purified 5-lipoxygenase such as molecular weight (74,000-76,000), N-terminal sequence (30 amino acids), and amino acid composition were determined. The purified enzyme converted [14C]arachidonic acid at 20 degrees to [14C] 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and to [14C]dihydroxyeicosatetraenoic acids (diHETEs). Utilizing [14C] 5(S)HPETE as substrate, the purified enzyme also converted the hydroperoxy acid to [14C]diHETES. The [14C]diHETE reaction products were identified primarily (greater than 80% of recovered radioactivity) as the nonenzymatic hydrolysis products of leukotriene A4 (i.e., 6-trans-leukotriene B4 and 12-epi-6-trans-leukotriene B4) by reverse phase HPLC, scanning spectrophotometry, and gas chromatography-mass spectrometry. The bioconversion of [14C] arachidonate and [14C]5(S)HPETE to reaction products by the purified enzyme was dependent on the presence of both Ca2+ and ATP. The enzymatic activities were inhibited in a similar manner by the lipoxygenase inhibitors nordihydroguaiaretic acid, diphenyldisulfide, and SK&F 86002. The data provide evidence that RBL-1 cell 5-lipoxygenase and leukotriene A4 synthetase activities reside on a single monomeric protein with a free N-terminus and that they possess similar biochemical characteristics.

Pure enzymes in a test hierarchy for antibronchoconstrictory lipoxygenase inhibitors.

A test hierarchy for potential antibronchoconstrictive drugs acting as lipoxygenase inhibitors was developed which includes a purified mammalian lipoxygenase/leukotriene A4 synthase, purified cyclooxygenase, arachidonic acid metabolism of polymorphonuclear leukocytes, arachidonic acid-induced contraction of lung strips, isolated human bronchi, and ovalbumin-induced bronchoconstriction with sensitized guinea pigs. The lipoxygenase from rabbit reticulocytes turned out to be superior to a variety of other animal and plant lipoxygenases. Among the various test systems with respiratory tract preparations, the arachidonic acid-induced contraction of guinea pig lung strips gave the most reliable results in the search for new antiasthmatic drugs. The validity of the test hierarchy was ascertained in screening investigations. Novel classes of antibronchoconstrictory lipoxygenase inhibitors were found.