Rat testis lipoxygenase-like enzyme characterization of products from linoleic acid

The linoleate oxidation products of the affinity chromatography-purified lipoxygenase-like enzyme isolated from rat testes microsomes were characterized. Three types of reaction products separated by thin-layer chromatography were generally present: polar byproducts (A and B) and hydroperoxides. The methyl hydroxystearates obtained from the enzymically produced hydroperoxides were analysed by gas-liquid chromatography and showed a ratio of 67% 13-hydroxy isomer to 33% 9-hydroxy isomer.The major polar byproduct was analysed by infrared spectra, nuclear magnetic resonance and mass spectrometry (of the toluene-p-sulphonyl derivative) and its structure was established as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid. The possibility of the existence of a linoleate hydroperoxide isomerase in the affinity-purified preparation is discussed.     

Aldehydic lipid peroxidation products derived from linoleic acid

Lipid peroxidation (LPO) processes observed in diseases connected with inflammation involve mainly linoleic acid. Its primary LPO products, 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) and 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), decompose in multistep degradation reactions. These reactions were investigated in model studies: decomposition of either 9-HPODE or 13-HPODE by Fe(2+) catalyzed air oxidation generates (with the exception of corresponding hydroxy and oxo derivatives) identical products in often nearly equal amounts, pointing to a common intermediate. Pairs of carbonyl compounds were recognized by reacting the oxidation mixtures with pentafluorobenzylhydroxylamine. Even if a pure lipid hydroperoxide is subjected to decomposition a great variety of products is generated, since primary products suffer further transformations. Therefore pure primarily decomposition products of HPODEs were exposed to stirring in air with or without addition of iron ions. Thus we observed that primary products containing the structural element R-CH=CH-CH=CH-CH=O add water and then they are cleaved by retroaldol reactions. 2,4-Decadienal is degraded in the absence of iron ions to 2-butenal, hexanal and 5-oxodecanal. Small amounts of buten-1,4-dial were also detected. Addition of m-chloroperbenzoic acid transforms 2,4-decadienal to 4-hydroxy-2-nonenal. 4,5-Epoxy-2-decenal, synthetically available by treatment of 2,4-decadienal with dimethyldioxirane, is hydrolyzed to 4,5-dihydroxy-2-decenal.     

Inability of skin enzyme preparations to biosynthesize arachidonic acid from linoleic acid

The lack of any information as to the origin of epidermal arachidonic acid, an important precursor of eicosanoids in the epidermis, prompted us to determine in vitro whether or not microsomal preparations from rat and guinea pig epidermis possess the delta 6 and delta 5 desaturase activities. The incubations were performed in parallel with microsomal preparations from liver of these animals where activities for these enzymes have previously been reported. The conversions of radioactive fatty acids were determined after methylation and separation of the 14C-fatty acid methyl esters by argentation thin layer chromatography. Data from these studies demonstrated that delta 5 desaturase activity is markedly lower in guinea pig liver than in rat liver. Interestingly, preparations from rat and guinea pig epidermis at all concentrations tested lacked the capacity to transform either linoleic acid into gammalinolenic acid or dihomogammalinolenic acid into arachidonic acid. This observation implies that arachidonic acid that is present in the epidermal phospholipids is biosynthesized elsewhere endogenously and transported to the epidermis for esterification into the phospholipids. The site of this biosynthesis is presumably the liver and the mode of transport to the epidermis remains to be determined. These studies indicate arachidonic acid per se as an essential fatty acid for the epidermis.     

Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis

Several lines of evidence indicate that abscisic acid (ABA) is derived from 9′-cis-neoxanthin or 9′-cis-violaxanthin with xanthoxin as an intermediate. ¹⁸O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11′, 12′) double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.     

Calcium-translocating and cardiotonic properties of oxidation products of linoleic acid

Calcium-translocating activity of linoleic acid and its lipoxygenase (linoleate: oxygen oxidoreductase; EC 1.13.11.12) metabolites or autoxidation products was determined in vitro by estimation of 45Ca transport from a bulk aqueous to a bulk organic phase. Fresh commercial linoleic acid, tested immediately after removal from a sealed vial, stimulated calcium translocation only at concentrations greater than 1 mM. In contrast, 45Ca translocation by linoleic acid exposed to air was detectable at 10 microM. Oxidation products of linoleic acid obtained either by incubation with lipoxygenase or by autoxidation were much less potent than the calcium ionophore A23187. The products obtained by enzymic oxidation of linoleic acid enhanced contractility in the Langendorff-perfused guinea pig heart up to 45% over control (at 3 X 10(-8) M). The inotropic response was transient with rapid onset and not affected by the beta-adrenergic antagonist, propranolol. The autoxidation products of linoleic acid increased cardiac contractility up to 43% at 10(-6) M. In contrast, fresh linoleic acid caused only a negative inotropic effect at 10(-8) to 3 X 10(-7) M, progressing to contracture at 10(-6) M. These findings suggest that conflicting reports on the cardiostimulant effect of linoleic acid may be due to varying levels of the autoxidation products. Linoleic acid metabolites in vivo may have a physiological role in myocardial function related to their Ca2+-ionophoric activity.     

Purification of acid beta-galactosidase and acid neuraminidase from bovine testis: evidence for an enzyme complex

The isolation of an acid neuraminidase from bovine testis is described. Under all experimental conditions this neuraminidase copurifies with acid β-galactosidase, but not with other lysosomal hydrolases. Immunotitration with an antiserum raised against purified human placental β-galactosidase results in the coprecipitation of both enzyme activities. Our data indicate that acid neuraminidase and β-galactosidase are present as an enzyme complex. The possible physiological relevance is discussed.     

Characterization of a soluble follitropin receptor from porcine testis.

Porcine testis receptors for follitropin (FSH) were solubilized by treatment with the non-ionic detergent Nonidet P-40 and receptor-bound and free ¹²⁵I-porcine FSH were separated by ammonium sulfate precipitation. The soluble receptor retained both its high affinity and specificity for FSH. The soluble hormone-receptor complex exhibited an equilibrium association constant of 4.7 × 10¹⁰ M^?1 at 4°C. Its hydrodynamic properties were consistent with those obtained for other solubilized peptide hormone receptors, and its molecular weight estimated to 244,000.     

Rat liver cytosolic glutathione peroxidase: reactivity with linoleic acid hydroperoxide and cumene hydroperoxide.

The reactivity of rat liver glutathione (GSH) peroxidase with two hydroperoxides was determined using integrated rate equations. The bimolecular rate constant for the reaction of GSH peroxidase with linoleic acid hydroperoxide is approximately four times the rate constant with cumene hydroperoxide. The reactivity toward reduced glutathione is not altered by different hydroperoxides. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for lipid hydroperoxide in rat liver is approximated at 9.5 × 10^?5 min.     

Profiling assay for lipoxygenase products of linoleic and arachidonic acid by gas chromatography-mass spectrometry.

A method for determination of the lipoxygenase products of linoleic acid (9- and 13-hydroxyoctadecadienoic acid; 9-HODE, 13-HODE) and of arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acid; 5-, 8-, 9-, 11-, 12-, and 15-HETE) is described. The method combines solid-phase extraction, derivatization to the corresponding fully hydrogenated methylester/trimethylsilylether derivatives and capillary gas chromatography coupled with electron impact mass spectrometry. Each regioisomeric HODE and HETE shows a unique pair of mass spectrometric fragment ions originating from fission of the fatty acid carbon chain at the hydroxylated position. The carboxyl-terminal fragment is used for quantification relative to a carboxyl-18O2-labeled analogue added as internal standard and the methyl-terminal fragment is monitored for confirmation. The assay can be extended for quantification of the complete hydroxylation profile of linoleic and arachidonic acid. Applications of this assay are demonstrated for the quantification of HODEs and HETEs in normal, hyperplastic, and neoplastic mouse epidermis. In mouse epidermis papilloma, the tissue levels of 8- and 12-HETE were found to be increased by one to two orders of magnitude compared to levels in normal epidermis.     

Stereochemical nature of the products of linoleic acid oxidation catalyzed by lipoxygenases from potato and soybean

When linoleic acid was incubated with the purified potato lipoxygenase under O2 atmosphere, a mixture of 9 and 13-hydroperoxyoctadecadienoic acids was formed. Stereochemical analysis of the respective methyl-hydroxyoctadecadienoic acids revealed that the 9-isomer was in S-configuration whereas 13-hydroxyoctadecadienoic acid was a mixture of S (39%) and R (61%). Exactly the opposite was the case with the soybean lipoxygenase products, where the 13-isomer was found to be in S-configuration and 9-hydroxyoctadecadienoic acid - a mixture of S (73%) and R (27%). A general scheme is proposed for the stereochemical nature of oxidation products of enzymes which are predominantly either [+2] or [-2] lipoxygenases.     

Reaction of linoleic acid hydroperoxide with thiobarbituric acid.

The linoleic acid hydroperoxide obtained by enzymatic peroxidation of linoleic acid was found to react with thiobarbituric acid to yield a red pigment. The optimum pH for the reaction was found to be 4.0. In the early stages of peroxidation of linoleic acid, thiobarbituric acid value, the amount of conjugated diene, oxygen consumption, and peroxide value were in parallel with one another. The data were compared with those on peroxidation of linolenic acid and arachidonic acid.     

Lipoxygenase-like enzyme in rat testis microsomes.

Microsomes, separated from rat testes, were found capable of oxidizing linoleate and arachidonate. The enzyme activity was solubilized with 1% Triton X-100 in acetate buffer (pH 5.0) and purified by affinity chromatography. The overall purification from the starting preparation was approx. 40-fold. The affinity-purified enzyme was almost homogeneous as determined by electrophoresis in polyacrylamide gel. The enzyme was characterized as lipoxygenase-like from its spectrum, specificity, effect of linoleate on its fluorescence and linoleate oxidation products. Three types of compounds separated by thin-layer chromatography were generally present in the lipoxygenase-like enzyme reaction on linoleic acid: substrate fatty acid, polar by-products and hydroperoxides. The hydroperoxides were analyzed by infrared spectra and mass spectrometry and showed the presence of both 9- and 13-hydroxy isomers.     

Lipoxygenase from potato tubers. Partial purification and properties of an enzyme that specifically oxygenates the 9-position of linoleic acid

A lipoxygenase (EC 1.13.1.13) was partially purified from potato tubers and was shown to differ from previously characterized soya-bean lipoxygenases in the positional specificity and pH characteristics of the oxygenation reaction. The potato enzyme converted linoleic acid almost exclusively (95%) into 9-d-hydroperoxyoctadeca-trans-10,cis-12-dienoic acid. The 13-hydroperoxy isomer was only a minor product (5%). Linolenic acid was an equally effective substrate, which was also oxygenated specifically at the 9-position. The enzyme had a pH optimum at 5.5-6.0 and was inactive at pH9.0. A half-maximal velocity was obtained at a linoleic acid concentration of 0.1mm. No inhibition was observed with EDTA (1mm) and cyanide (1mm) or with p-chloromercuribenzoate (0.2mm). Haemoproteins were not involved in the lipoxygenase activity. The molecular weight of the enzyme was estimated from gel filtration to be approx. 10(5). Preliminary evidence suggested that the enzyme oxygenated the n-10 position of fatty acids containing a penta(n-3, n-6)diene structure.     

Phase 2 enzyme induction by conjugated linoleic acid improves lupus-associated oxidative stress

Conjugated linoleic acid (CLA) exhibits anticancer and anti-inflammatory properties. Its ability to increase total GSH (GSH+GSSG) amount and gamma-glutamylcysteine ligase (gammaGCL) protein expression was recently associated with the inhibition of typical pathological signs in MRL/MpJ-Fas(lpr) mice (MRL/lpr). In the present study the ability of CLA to modulate oxidative stress and phase 2 enzyme activity in the same animal model was investigated. Disease severity was associated with age-dependent production of anti-double-stranded DNA antibodies (anti-dsDNA IgGs) and with enhanced extent of oxidative stress markers: reduced total GSH, increased protein 3-nitrotyrosines (3-NT), and protein-bound carbonyl (PC) amounts. To examine the effect of CLA on antioxidant status, CLA or olive oil (as control) was administered to pregnant MRL/lpr mice. Significantly higher total GSH and Trolox equivalent antioxidant capacity (TEAC) levels were measured in serum of CLA-treated dams (and their pups), as compared with controls. Finally, the antioxidant and chemopreventive properties of CLA were investigated in old MRL/lpr mice. Sera of CLA-treated mice contained higher concentrations of total GSH which were negatively correlated with the levels of oxidative stress markers. Moreover, increased GSH, gammaGCL, glutathione S-transferase (GSTs), and NAD(P)H:quinone oxidoreductase (NQO1) activities were measured in liver and spleen of CLA-treated animals. In conclusion our data indicate that the activation of detoxifying enzymes may be one of the mechanisms whereby dietary CLA down-regulates oxidative stress in MRL/lpr mice.     

Bioconversion of linoleic acid to conjugated linoleic acid byBifidobacterium breve

The bioconversion of linoleic acid (LA) to conjugated linoleic acid (CLA) was investigated to examine LA-adaptation ofBifidobacterium breve KCTC 3461 to additions of 1 to 5 mg/mL of LA overtime. To induce LA-adaptation,B. breve KCTC 3461 was treated with LA, according to three schemes. For LA-adaptedB. breve the maximum concentration of CLA, 300–350 μg/mL, was obtained in cys-MRS medium containing 1 mg/mL of LA. The CLA production significantly increased with increasing LA concentration, from 1 to 4 mg/mL, but the conversion of LA to CLA gradually decreased. The CLA production capability ofB. breve, and its tolerance, improved significantly with LA-adaptation. The addition of LA (1 mg/mL) into the culture broth after 24 h of cultivation in a 100-mL media bottle was most effective at promoting CLA production. In a 2.5-L stirred-tank bioreactor, the observed conversion and productivity of 56.6% and 35.4 μgml⁻¹h⁻¹, respectively, by LA-adaptedB. breve were approximately 6.6 and 9.8 times higher than those of LA-unadaptedB. breve.     

Rat testis immunoreactive LH-RH differs structurally from hypothalamic LH-RH

Luteinizing hormone releasing hormone immunoreactivity (LH-RH-IR) has been identified in acetic acid extracts of adult rat testes and partially purified by immunoaffinity chromatography. On Sephadex G-100 this material separated into four major peaks of >100K, ～32K, ～5K and ≤4K daltons. The ≤4K peak of LH-RH-IR eluted later than synthetic hypothalamic LH-RH decapeptide on Sephadex G-25. Antibody binding studies on the various LH-RH-IR species with antisera specific for different regions of synthetic LH-RH decapeptide indicate that all the testicular LH-RH-IR molecules have C-terminal immunological homology with the hypothalmic decapeptide but differ towards the N-terminus of the decapeptide sequence.