Porcine leukocyte 5- and 12-lipoxygenases are iron enzymes.

5- and 12-lipoxygenases isolated from porcine leukocytes were investigated by electron paramagnetic resonance at X-band and atomic absorption spectroscopy. For comparison potato 5-lipoxygenase was studied under identical experimental conditions. All three lipoxygenases contained between 0.7 and 0.9 Fe atoms/enzyme molecule. As isolated, both mammalian enzymes exhibited a characteristic EPR signal at low magnetic field with a maximum at g = 5.20 indicative of a high-spin ferric iron center. The signal was not affected by the oxidants 12-hydroperoxyeicosatetraenoic acid or arachidonic acid, nor was it affected by the reductant nordihydroguaiaretic acid. In the case of the potato enzyme an intense EPR signal with resonances at g = 7.50, 6.39 and 5.84 was only observed after addition of an oxidant, such as 9-hydroperoxyoctadecadienoic acid.

Ebselen affects calcium homeostasis in human platelets.

Ebselen (PZ 51, 2-phenyl-1,2-benzoisoselenazol-3-(2H)-one) is a selenoorganic compound with anti-inflammatory properties. Its pharmacological action is thought to originate from its peroxidase activity which could lower the peroxide tonus required for cyclooxygenase and lipoxygenase activations. From experiments with aspirin-treated human platelets we now present evidence that ebselen also affects intracellular calcium homeostasis by inhibiting the agonist-triggered increase in intracellular calcium. Using Mn2+ entry to quench the fura-2 fluorescence after cell stimulation, we could exclude an interaction of ebselen with receptor-operated calcium channels and therefore an inhibition of extracellular calcium influx. It became evident from whole cell experiments and by using isolated platelet microsomal vesicles that ebselen inhibits the inositol 1,4,5-trisphosphate (IP3) induced calcium release. Besides this inhibitory effect of ebselen on the calcium release higher concentrations of the compound (greater than or equal to 5 microM) induced a calcium release from our microsomal vesicles which also could be reversed by dithiothreitol. An activation of inflammatory cells is usually associated with increased cytosolic calcium concentrations. An inhibition of such calcium movements by ebselen may account for an up to now unidentified anti-inflammatory mechanism of ebselen action which is linked to a direct effect of this compound rather than to its peroxidase-like activity.

12(S)-Hydroxy-5,8,10 (Z,E,E)-heptadecatrienoic acid (HHT) is preferentially metabolized to its 12-keto derivative by human erythrocytes in vitro.

The metabolism of [1-14C]-labelled 12 (S)-hydroxy-5,8,10 (Z,E,E)-heptadecatrienoic acid (HHT) by crude 15-hydroxyprostaglandin dehydrogenase (PGDH) fractions from swine kidney and human erythrocytes has been investigated. HPLC radiochromatography analysis revealed that HHT was extensively converted into three metabolites by swine kidney cytosol in the presence of NAD+. They were identified by combined GLC mass spectrometry as 12-keto-5,8,10 (Z,E,E)-heptadecatrienoic acid (KHT), 12-keto-5,8 (Z,E)-heptadecadienoic acid and 12 (RS)-hydroxy-5,8 (Z,E)-heptadecadienoic acid, respectively. In contrast, HHT was metabolized only to the 12-keto derivative by human erythrocyte cytosol supplemented with NADP+, and HHT turnover was found to be enhanced severalfold when compared to prostaglandins E2 (PGE2) or F2 alpha. Since PGE2 was also converted only into 15-keto-PGE2, and no metabolism of KHT was detected with NADPH, there is probably no 15-ketoprostaglandin delta 13-reductase activity in human erythrocytes. Biosynthetic KHT (0.5-5 microM) inhibited the aggregation of human platelets to almost all agonists, probably by raising intracellular cAMP. KHT (between 0.01 and 1 microM) also induced the chemotaxis of human polymorphonuclear leukocytes. Among other still unrecognized effects, these biological activities of KHT may be of physiological significance with respect to its presumably exclusive formation in the blood. The potential use of KHT for monitoring thromboxane synthase activity in vivo is discussed.

Thromboxane synthase catalyses hydroxylations of prostaglandin H2 analogs in the presence of iodosylbenzene.

Human platelet microsomes supplemented with iodosylbenzene converted the stable prostaglandin H2 analog 15(S)-hydroxy-11 alpha,9 alpha-epoxymethano-5(Z),13(E)-prostadienoic acid (U46619) into three metabolites (17.5% yield) which were not formed in the presence of specific thromboxane synthase inhibitors. The same three products were also formed among others by incubation of U46619 with liver microsomes from phenobarbital-pretreated rats with NADPH/O2 or with iodosylbenzene. The NADPH-supported metabolism of U46619 was suppressed in the presence of carbon monoxide. Combined gas chromatography/negative-ion chemical ionization mass spectrometry analysis revealed for all three compounds the incorporation of one oxygen atom which according to the electron impact fragmentation pattern had to be introduced either at the 9-methylene group or at the cyclopentane ring. The identification of these metabolites as 9 beta,15(S)-dihydroxy-11 alpha,9 alpha-epoxymethano-5(Z),13(E)-prostadienoic acid and the R and S isomer of 15(S)-hydroxy-11 alpha,9 alpha-(C-hydroxy-epoxymethano)-5(Z),13(E)-prostadienoic acid is only tentative since no reference compounds were available, but clearly thromboxane synthase was acting as an oxene transferase in this reaction. In contrast to U46619, its 9,11-epoxymethano isomer U44069 was found to be only a poor substrate for the oxene transferase activity of thromboxane synthase (1% yield) which indicates a preference for the 9-methylene group of U46619 which is orientated close to the heme iron of thromboxane synthase as evidenced by spectroscopic studies. Low-level chemiluminescence detected following incubation of iodosylbenzene with partly purified thromboxane synthase is in agreement with the formation of an activated (FeO)3+ oxygen species. In summary, these results point to a common role of the thiolate ligand in the oxygen activation mechanism by thromboxane and prostacyclin synthase and liver cytochrome P-450 monooxygenases.

Spectral intermediates of prostaglandin hydroperoxidase.

Microsomes from ram seminal vesicles or purified prostaglandin H synthase supplemented with either arachidonic acid or prostaglandin G2 formed an unstable spectral intermediate with maxima at 430 nm, 525 nm and 555 nm and minima at 410 nm, 490 nm and 630 nm. At -15 degrees C the band at 430 nm disappeared within 4 min whereas the trough at 410 nm increased three fold. At higher temperatures (10-37 degrees C) spectral complex formation and decay were observed in less than 1 s. An apparent KS-value of about 3 microM was determined for the titration of purified prostaglandin synthase with prostaglandin G2 at -20 degrees C. Substrates for cooxidation reactions of prostaglandin synthase such as phenol, hydroquinone and reduced glutathione as well as the peroxidase inhibitors cyanide and azide inhibited the prostaglandin G2-induced spectral complex formation. The oxene donor iodosobenzene and hydrogen peroxide formed a spectral intermediate analogous to the complex observed with prostaglandin G2 or arachidonic acid in ram seminal vesicle microsomes as well as with the purified prostaglandin synthase. These results are interpreted as the formation of a ferryl-oxo complex (FeO)3+ of the heme of prostaglandin synthase with prostaglandin G2 analogous to the formation of compound I of horseradish peroxidase.

A quantitative test for superoxide radicals produced in biological systems.

The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH--cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced in biological membranes, e.g. microsomes, are discussed.