Ovothiol: a novel thiohistidine compound from sea urchin eggs that confers NAD(P)H-O2 oxidoreductase activity on ovoperoxidase

Sea urchin eggs contain a small molecular weight heat-stable factor that confers cyanide-resistant NAD(P)H-O2 oxidoreductase activity on ovoperoxidase (Turner, E., Somers, C. E., and Shapiro, B. M. (1985) J. Biol. Chem. 260, 13163-13171), the enzyme responsible for cross-linking the extracellular protein coat (fertilization membrane) of the egg. Here we report the isolation of the active cofactor and its identification by ultraviolet, NMR, and mass spectroscopy as a new sulfur-containing amino acid derivative, 1-methyl-alpha N,alpha N-dimethyl-4-thiohistidine, or ovothiol. Ovothiol reacts slowly with atmospheric oxygen or rapidly with micromolar concentrations of H2O2 to form ovothiol disulfide, which is inactive as a cofactor for the ovoperoxidase NAD(P)H oxidase. Reduced active ovothiol is regenerated by treatment with disulfide reductants and shows significant differences in its ultraviolet and NMR spectra from oxidized ovothiol. The oxidoreductase activity of the ovoperoxidase/ovothiol system is similar to that previously characterized with crude cofactor preparations; it is greatly enhanced by Mn2+ and is relatively insensitive to CN-, compared to the peroxidase activity of ovoperoxidase. The ovothiol content of eggs is estimated as 1.8 pmol/egg or an intracellular concentration of 6.8 mM. This concentration exceeds the amount of reductant needed for the CN-(-)insensitive oxygen consumption following fertilization and used in the production of H2O2 for fertilization membrane cross-linking. Whether ovothiol is involved in the cross-linking reaction, protects the egg from damage from H2O2, or has another role in development remains unclear.     

L-1-N-methyl-4-mercaptohistidine disulfide, a potential endogenous regulator in the redox control of chloroplast coupling factor 1 in Dunaliella.

A water-soluble, highly polar, heat-stable, small molecule has been isolated from cell-free extracts of the halotolerant green alga Dunaliella salina. This compound, soluble inhibitory factor (SIF), when added to in vivo light-activated, thylakoid membrane-bound preparations of the D. salina coupling factor 1 (CF1), causes a rapid inactivation of the ATPase activity. SIF must be in its oxidized form to inactivate the CF1 ATPase and probably functions by oxidizing the reduced form of the light-activated enzyme. SIF has been purified to homogeneity and characterized by UV-visible and IR absorption spectroscopy, 1H and 13C NMR spectroscopy, and mass spectrometry. SIF has five different kinds of nonexchangeable protons and seven different kinds of carbon atoms. Three of the carbon atoms and one proton are part of a heterocyclic (imidazole) ring. One carbon atom is a carbonyl (carboxylic acid). One carbon atom and three protons form a methyl group attached to the aromatic ring. One carbon atom and two protons are a methylene group, and one carbon atom (an alpha-amino carbon) is attached to a single proton. In addition, in its reduced form, SIF contains a thiol group attached to the heterocyclic ring. From high resolution mass spectrometry, the molecular weight of SIF was determined to be 401 (M + H+) and is consistent with the composition being C14H21N6O4S2. The UV absorption of SIF shows a large increase at 240 nm upon reduction. An effective difference extinction coefficient for this absorbance change has been calculated to be 6.84 meq/cm. A comparison of SIF with the oxidized form of ovothiol A (1-N-methyl-4-mercaptohistidine disulfide) shows the two compounds to be identical in all respects. In addition, ovothiol A disulfide is as effective as SIF in inhibiting the light-triggered, CF1 ATPase activity. It is concluded, therefore, that SIF and L-1-N-methyl-4-mercaptohistidine disulfide are identical.     

Identification of a naturally occurring methyl-ester of phosphate, methyl-phosphorylcholine (methyl-2-(N,N,N trimethylamino) ethyl phosphate), in the eggs of the sea urchin S. purpuratus

A novel metabolite of choline, phosphorylcholine methyl ester, has been identified in the eggs of S. purpuratus wherein it is present at approximately 1 mM concentration. To the best of our knowledge, this is the first instance of a phosphoryl-methyl-ester to be observed in nature. The compound appears to be species specific, since it has not been observed in other species such as L. pictus and P. depressus. In S. purpuratus its distribution is confined to the ovary, eggs and embryos, and is absent from young animals following metamorphosis.     

Oxidative stress and the role of novel thiol compounds at fertilization

A new class of thiols, the 1-methyl-4-mercaptohistidines, has been found in high concentrations in invertebrate eggs. This family, called the ovothiols, has unusual redox properties, including the ability to confer a CN- -resistant NAD(P)H oxidase activity on ovoperoxidase, the enzyme that catalyzes the physiological crosslinking of the fertilization envelope with dityrosine residues. Ovothiol has a redox potential of 44 mV positive to glutathione and thus is maintained in the reduced state in eggs by reduced glutathione, without the need for an ovothiol reductase. We propose that high concentrations of reduced ovothiol are present in eggs to protect them from the oxidative stress caused by the respiratory burst of fertilization.     

Ovothiols as free-radical scavengers and the mechanism of ovothiol-promoted NAD(P)H-O2 oxidoreductase activity

Racemic ovothiol A [(+/-)-1a] and the ovothiol model compound 1,5-dimethyl-4-mercaptoimidazole (DMI, 2) were found to scavange the free radicals Fremy's salt (4) and Banfield' radical (5) much more rapidly than did the thiol antioxidant glutathione. Ovothiol A also scavenges the tyrosyl radical, with efficiency comparable to that of ascorbic acid and the tocopherol analogue trolox (3). The ovothiol model compound DMI was found to scavenge superoxide with a rate constant comparable to that of the reaction between superoxide and glutathione. These results suggest both a free-radical scavenging role for the ovothiols and a mechanism by which the ovothiols confer NAD(P)H-O2 oxidoreductase activity upon the enzyme ovoperoxidase. Investigation of this mechanism implicates the ovothiol thiyl radical and the NAD radical as key intermediates. The ovothiyl radical appears to be unreactive toward oxygen but highly reactive toward NADH. An estimate of the one-electron oxidation potential of the ovothiol anion is presented. The physical basis for the stability of the ovothiol free radical is discussed.     

The biosynthesis of ovothiol A (N-methyl-4-mercaptohistidine). Identification of S-(4'-L-histidyl)-L-cysteine sulfoxide as an intermediate and the products of the sulfoxide lyase reaction.

Crude extracts of Crithidia fasciculata catalyse the formation of 4-mercapto-L-histidine, an intermediate in the biosynthesis of ovothiol A (N1-methyl-4-mercaptohistidine), in the presence of histidine, cysteine, Fe2+ and pyridoxal phosphate. This activity was present in a 35-55% ammonium sulfate fraction that was shown to produce a transsulfuration intermediate in the absence of pyridoxal phosphate. The transsulfuration intermediate was isolated and identified as S-(4'-L-histidyl)-L-cysteine sulfoxide. The synthase activity, partially purified by anion-exchange chromatography, was shown to require oxygen and could be used to synthesize a number of isotopically labeled S-(4'-L-histidyl)-L-cysteine sulfoxides. Sulfoxide lyase activity was partially resolved from the synthase by anion-exchange chromatography. The phenylhydrazone of the product derived from the cysteine moiety of the sulfoxide coeluted with the phenylhydrazone of pyruvate on HPLC, but this assignment could not be confirmed by mass spectral analysis. S-(4'-[14C]L-histidyl)-[U-13C3,15N]L-cysteine sulfoxide was synthesized and converted to products of the lyase reaction in the presence of lactate dehydrogenase and NADH. The 13C-labeled product was identified by 13C-NMR spectroscopy as lactate and the primary product of the lyase reaction is therefore pyruvate. With S-(4'[3H]L-histidyl)-[14C]L-cysteine sulfoxide as the substrate [14C]lactate, [14C]cysteine and [3H]4-mercaptohistidine could be detected as products of the lyase reaction, but the sum of the two thiol species exceeded the amount of sulfoxide substrate used. Evidence is presented that this anomaly was due to the utilization of sulfur from dithiothreitol for the formation of cysteine.     

Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase

A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.     

黑柄炭角菌产生的DPPH自由基捕捉成分

对黑柄炭角菌深层发酵制品中的DPPH自由基捕捉成分进行研究。经硅胶柱层析、中压液相色谱顺相和反相分离、制备型高压液相色谱分离等一系列步骤 ,共获得相对纯度在85%以上 ,收量在 2mg以上的自由基捕捉物质 2 0个 ,对其中的B4 1 6进行了质谱、1H NMR、13C NMR、1H 13CHMBC、红外光谱等的测定 ,测得分子式为C10 H10 O4 ,推断它为 5,8二羟基 3 甲基 3,4二氢异香豆素。在 2 0 μmol L时 ,它的DPPH自由基捕捉活性为维生素C的 1 67倍 ,维生素E的 2 1倍。     

Glutamyl ribose 5-phosphate storage disease. A hereditary defect in the degradation of poly(ADP-ribosylated) proteins

A patient with a lysosomal storage disease, progressive neurologic degeneration, and renal failure was found to have accumulated a low molecular weight ninhydrin and phenol-H2SO4 reactive compound. Amino acid analysis and gas chromatography-mass spectrometry identified a glutamic acid moiety. Direct insertion mass spectrometry proved the carbohydrate portion to be a sugar phosphate. NaB3H4 reduction and borate electrophoresis, paper chromatography, and enzymatic digestion indicated the presence of ribose 5-phosphate. Quantitative analysis of the intact compound indicated a 1:1:1 ratio for glutamic acid: ribose:phosphate. Brain was found to contain 0.96 mumol/g, wet weight, and kidney 0.60 mumol/g, wet weight, of glutamyl ribose 5-phosphate. This substance is the linkage region in ADP-ribosylation of histones and other proteins. It is suggested that the primary defect in this patient is a genetic abnormality of ADP-ribose protein hydrolase (Okayama, H., Honda, M., and Hayaishi, O. (1978) Proc. Natl. Acad. Sci. U. S .A. 75, 2254-2257).     

1-Methyl-4-thiohistidine and Glutathione in the Developing Embryo of the Sea Urchin, Paracentrotus lividus.

4-thiohistidines occur as free amino acids in the eggs of several marine organisms, in particulary relevant concentrations in the eggs of echinoderms. Since nothing is known about their biological role or biosynthesis and metabolic fate, we measured the concentration of 1-methyl-4-thiohistidine together with the two most common small molecular weight thiols, glutathione and cysteine, during embryonic development of the sea urchin Paracentrotus lividus until the pluteus stage. The thiols were determined by high performance liquid chromatography as fluorescent derivatives of monobromobimanes. 1-methyl-4-thiohistidine and glutathione are present until the pluteus stage with an approximate ratio of 2: 1. Cysteine was detected at the blastula stage and found to increase thereafter. 1-methyl-4-thiohistidine was absent, or present in traces, in sperm and in somatic tissues of adults. It is concluded that 1-methyl-4-thiohistidine is typical of eggs and embryos and may be metabolized during metamorphosis into an as yet unknown compound.     

Phosphorylation on protein and carbohydrate moieties of a lysosomal arylsulfatase B variant in human lung cancer transplanted into athymic mice

Human lung cancer transplanted into athymic mice contains predominantly an acidic variant (designated B1) of lysosomal arylsulfatase B. B1 enzyme was suggested to be phosphorylated and sialylated (Gasa, S., Makita, A., Kameya, T., Kodama, T., Koide, T., Tsumuraya, M., and Komai, T. (1981) Eur. J. Biochem. 116, 497-503). In order to determine the localization of phosphate in B1 enzyme, we labeled in vivo the transplanted tumor with [32P]H3PO4 or [3H]glucosamine and purified B1 enzyme by immunoprecipitation. Bio-Gel chromatography of the labeled B1 enzyme treated with endoglycosidase H demonstrated that both the excluded and included materials were labeled with 32P and 3H. From acid hydrolysate of the excluded materials, phosphorylated serine and threonine were detected. Protein phosphorylation of arylsulfatase was confirmed by in vitro labeling experiments with [gamma-32P]ATP. By incubation of the tumor homogenate with ATP followed by isolation of the enzymes, B1 enzyme had a significant amount of radioactivity, whereas the B enzyme had little; by exogenous protein kinase, partially purified B enzyme was phosphorylated 35 times more than B1 enzyme. Acid hydrolysate of the included materials in the Bio-Gel column demonstrated mannose 6-phosphate and an unknown phosphorylated compound which migrates more than Man-6-P on electrophoresis and chromatography.     

Kinetics of actin assembly attending fertilization or artificial activation of sea urchin eggs

Changes in the state of actin assembly triggered by fertilization or by artificial activation of sea urchin eggs were quantified using the DNase I inhibition assay. Insemination of Lytechinus pictus or Strongylocentrotus purpuratus eggs induces a cyclic variation in the level of G-actin as follows: between 0 and 30 s after insemination, the G-actin content decreases. This is followed by an increase in the amount of monomeric actin between 30 and 60 s, and then from 60 s to 5 min postinsemination there is a progressive decrease in the egg's level of G-actin. This latter decrease is more pronounced in S. purpuratus eggs than in L. pictus eggs. Using sperm mimetics that trigger an increase in intracellular calcium concentration (A23187 in sodium-free seawater), a cytoplasmic alkalinization (NH4Cl), a plasma membrane depolarization (seawater enriched with potassium ions), or all three of these phenomena (A23187 in normal seawater), each phase depicted at fertilization correlates with the following metabolic events accompanying egg awakening: phase 1, of uncertain origin (possibly related to plasma membrane depolarization); phase 2, elevation of intracellular calcium concentration; phase 3, alkalinization of the intracellular milieu but only if the transient intracellular calcium rise has taken place.     

Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization

Egg activation at fertilization requires the release of Ca(2+) from the egg's endoplasmic reticulum, and recent evidence has indicated that a Src family kinase (SFK) may function in initiating this signaling pathway in echinoderm eggs. Here, we identify and characterize a SFK from the sea urchin Strongylocentrotus purpuratus, SpSFK1. SpSFK1 RNA is present in eggs, and an antibody made against a SpSFK1 peptide recognizes an approximately 58-kDa egg membrane-associated protein in eggs of S. purpuratus as well as another sea urchin Lytechinus variegatus. Injection of both species of sea urchin eggs with dominant-interfering Src homology 2 domains of SpSFK1 delays and reduces the release of Ca(2+) at fertilization. Injection of an antibody against SpSFK1 into S. purpuratus eggs also causes a small increase in the delay between sperm-egg fusion and Ca(2+) release. In contrast, when injected into eggs of L. variegatus, this same antibody has a dramatic stimulatory effect: it causes PLCgamma-dependent Ca(2+) release like that occurring at fertilization. Correspondingly, in lysates of L. variegatus eggs, but not S. purpuratus eggs, the antibody stimulates SFK activity. Injection of L. variegatus eggs with another antibody that recognizes the L. variegatus egg SFK also causes PLCgamma-dependent Ca(2+) release like that at fertilization. These results indicate that activation of a Src family kinase present in sea urchin eggs is necessary to cause Ca(2+) release at fertilization and is capable of stimulating Ca(2+) release in the unfertilized egg via PLCgamma, as at fertilization.     

Immunolocalization of hyalin in sea urchin eggs and embryos using an antihyalin-specific monoclonal antibody.

Monoclonal antibodies were raised against purified cortical secretory vesicles (CVs) from the eggs of Strongylocentrotus purpuratus. One of the monoclonal antibodies (MAb 69-10, an IgA) was shown by immunofluorescence labeling of intact and detergent-lysed CVs to be directed against a CV content antigen. Immunoblot analysis of CVs revealed that MAb 69-10 bound to a major CV polypeptide with an Mr similar to that of hyalin (i.e., 300,000). MAb 69-10 was subsequently shown to bind to purified hyalin prepared from S. purpuratus and to cross react with hyalin prepared from Lytechinus pictus. Immunogold labeling on thin sections of unfertilized S. purpuratus eggs showed that hyalin was localized to the electron-lucent portion of CVs. This result is in agreement with the labeling pattern obtained by Hylander and Summers (Dev Biol 93:368-380, 1982) using polyclonal antihyalin antibodies. In fertilized eggs and later-stage embryos, hyalin was observed to be located on the external surface of the embryo. MAb 69-10 should be useful in studies of the structure of hyalin and its function in morphogenesis.     

Protein tyrosine kinase activity of eggs of the sea urchin Strongylocentrotus purpuratus: the regulation of its increase after fertilization

Protein tyrosine kinase activity in eggs of the sea urchin, Strongylocentrotus purpuratus, increased two- to fourfold as early as several min after fertilization at 8-10 degrees C. Artificial activation of eggs with the divalent cation ionophore, A23187, or with butyric acid induced the increase in enzyme activity. The transfer of eggs to seawater containing either no Na+ or 50 mM Na+ and 10(-4) M amiloride immediately after fertilization did not block the increases in enzyme activity. When eggs were activated with seawater containing NH4OH, enzyme activity did not increase at 1 hr after activation, although the increased activity was detected at 3 hr after activation. Increased enzyme activity also was observed in enucleated egg fragments activated with butyric acid. Puromycin and emetine, inhibitors of protein synthesis, also did not inhibit the initial increases of enzyme activity after fertilization. These results demonstrated that the increased protein tyrosine kinase activity observed after fertilization of S. purpuratus eggs can be initiated independent of various other known events such as fusion with sperm cells and protein and DNA synthesis.     

The effect of buthionine sulfoximine on the growth of Leishmania donovani in culture

Changes in composition of the principal low molecular mass thiols of Leishmania donovani were monitored during the transformation of promastigotes, first to stationary phase metacyclic forms and then to amastigotes. No consistent variation in the thiol composition of the parasite which could account for the known increase in resistance of metacyclic and amastigote lifecycle forms to oxidant stress could be established. Amastigotes cultivated at 37 degrees C also produced ovothiol A, as judged by incorporation of radiolabel from [3-methyl]methionine and [14C]histidine, and the incorporation of radiolabel from [35S]cysteine into ovothiol A represented about 10-15% of the total label recovered in ovothiol A, glutathione and trypanothione. Amastigotes were less susceptible than promastigotes to the effects of the redox cyclers paraquat and menadione and grew in culture in the presence of up to 20 mM buthionine sulfoximine, which completely blocked the synthesis of glutathione and its spermidine conjugates. Glutathione and trypanothione biosynthesis is, therefore, not necessary for the replication of L. donovani amastigotes in culture. Inhibition of the formation of glutathione and trypanothione did not result in an upregulation of ovothiol A production.     

Effects of trialkyllead compounds on growth, respiration and ion transport in Escherichia coli K12

Triethyllead and tripropyllead cations affected growth, energy metabolism and ion transport in Escherichia coli K12. The tripropyllead compound was more liposoluble than the triethyl analogue and was also more effective in inhibiting cell growth and the oxygen uptake of both intact cells and membrane particles. Triethyllead acetate (5 microM) inhibited growth on non-fermentable carbon sources, such as glycerol and succinate, more markedly than on glucose. At higher concentrations, triethyllead caused significant inhibition of respiration rates of intact cells; the concentration giving 50% inhibition was 60 microM for glycerol-grown cells and 150 microM for glucose-grown cells. Oxidation of succinate by membrane particles was less sensitive to inhibition by the tripropyl- or triethyllead compounds than were the oxidations of DL-lactate or NADH. Triethyllead acetate [1.9 mumol (mg membrane protein)-1] inhibited the reduction by NADH of cytochromes; evidence for more than one site of inhibition in the respiratory chain was obtained. Membrane-bound ATPase activity was strongly inhibited by triethyllead acetate in the absence or presence of Cl-. The concentration of inhibitor giving 50% inhibition [0.02 mumol (mg membrane protein)-1] was about two orders of magnitude lower than that required for 50% inhibition of substrate oxidation rates in membranes. Triethyllead acetate (1 microM) induced swelling of spheroplasts in iso-osmotic solutions of either NH4Cl or NH4Br, presumably as a result of the mediation by the organolead compound of Cl-/OH- and Br-/OH- antiports across the cytoplasmic membrane. Similar exchanges of OH- for F-, NO3- or SO4(2)- or the uniport of H+ could not be demonstrated. Comparisons are drawn between the effects of trialkyllead compounds and those of the more widely studied trialkyltin compounds.     

Substrate specificity of formylglycinamidine synthetase

Formylglycinamidine ribonucleotide (FGAM) synthetase, which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and ATP to FGAM, ADP, glutamate, and Pi, has been purified to homogeneity (sp act. 0.20 mumol min-1 mg-1) from chicken liver by an alternative procedure to that of Buchanan et al. [Buchanan, J. M., Ohnoki, S., & Hong, B. S. (1978) Methods Enzymol. 51, 193-201] (sp act. 0.12 mumol min-1 mg-1). A variety of new analogues of formylglycinamide ribonucleotide have been prepared in which the formylglycinamide arm (R = CH2NHCHO) has been replaced by R = CH3, CH2OH, CH2Cl, CH2NH3, CH2NHCOCH3, CH2NHCOCH2Cl, CH2NHCO2CH2Ph, and L-CHC-H3NHCHO. These compounds have been characterized by 1H and 13C NMR spectroscopy. With compounds R = CH3, CH2OH, and CH2NHCOCH3 and ATP, in the presence or absence of glutamine, FGAM synthetase catalyzes the production of Pi at 4.5, 48, and 20%, respectively, the rate of production of Pi from formylglycinamide ribonucleotide. Only R = CH2NHCOCH3 causes glutaminase activity as well as ATPase activity and has been shown to be converted to the amidine analogue. Both FGAR (R = CH2NHCHO) and the FGAR analogue (R = CH2NHCHOCH3) in the presence of ATP and FGAM synthetase and in the absence of glutamine form a complex isolable by Sephadex G-50 chromatography. FGAM synthetase is thus highly specific for its formylglycine side chain. [18O]-beta-FGAR was prepared biosynthetically, and FGAM synthetase was shown by 31P NMR spectroscopy to catalyze the transfer of amide 18O to inorganic phosphate.     

Eggs of the sea urchin, Strongylocentrotus purpuratus, contain a prominent (11R) and (12R) lipoxygenase activity

Recent work has shown that oocytes of the starfish synthesize (8R)-hydroxyeicosatetraenoic acid and that this eicosanoid has a potent and highly specific action in induction of oocyte maturation. These striking results prompted us to examine the lipoxygenase activity of eggs of the sea urchin Strongylocentrotus purpuratus. Four hydroxyeicosanoids were formed in homogenates of sea urchin eggs; their structures and stereochemistry were characterized by high pressure liquid chromatography, UV spectroscopy, and gas chromatography-mass spectrometry. The compounds were identified as (11R)-hydroxy-5,8,12,14-ZZEZ-eicosatetraenoic acid and (12R)-hydroxy-5,8,10,14-ZZEZ-eicosatetraenoic acid (from arachidonic acid) and the corresponding (11R)- and (12R)-hydroxy analogs of eicosapentaenoic acid. The formation of these egg products was not blocked by a cyclooxygenase inhibitor, indomethacin (10 microM), and their precise structures are consistent with their formation by a lipoxygenase reaction. Eicosapentaenoic acids with a prochiral tritium label in the 10-D or 10-L position were used to investigate the mechanism of biosynthesis. The formation of (12R)-hydroxyeicosapentaenoic acid proceeded with the stereoselective abstraction of the 10-D hydrogen from the substrate. This reaction was shown to be opposite to the (12S) oxygenation catalyzed by porcine leukocyte 12-lipoxygenase. These results with S. purpuratus eggs constitute the first demonstration of (11R)- or (12R)-lipoxygenase activity in any cell type or tissue.     

Bioactive saponins from Astragalus suberi L. growing in Yemen

From the aerial parts of Astragalus suberi L., Fabaceae, seven saponins were isolated. Based on spectral data (IR, 1H and 13C NMR and HR-FABMS), the structures were established as 3-O-(beta-D-glucopyranosyl)-soyasapogenol B (1); 3-O-(beta-D-glucuronopyranosyl)-soyasapogenol B (2); 3-O-[beta-D-galactopyranosyl (1-->2)-beta-D-glucopyranosyl]-soyasapogenol B (3); 3-O-[alpha-L-rhamnopyranosyl (1-->2)-beta-D-galactopyranosyl (1-->2)-beta-D-glucopyranosyl]-soyasapogenol B (4); 3-O-[beta-L-rhamnopyranosyl (1-->2)-beta-D-galactopyranosyl (1-->2)-beta-D-glucopyranosyl]-11-hydroxy-soyasapogenol B (5); 3-O-[alpha-L-rhamnopyranosyl (1-->2)-beta-D-galactopyranosyl (1-->2)-beta-D-glucuronopyranosyl]-soyasapogenol B (6) and 3-O-[alpha-L-rhamnopyranosyl (1-->2)-beta-D-galactopyranosyl (1-->2)-beta-D-glucuronopyranosyl]-complogenin (7). The isolated saponins exhibited antibacterial activities against Gram-positive and Gram-negative bacteria with minimum inhibitory concentration values >100 microg/ml, antifungal activity against all the strains tested with minimum fungicidal concentration values between 25 and 50 microg/ml and inhibited the growth of Hep-2 (human carcinoma of larynx), with IC50 values between 50 microg/ml (compounds 5-7) and 100 microg/ml (compounds 1-4), and Hela (human carcinoma of cervix) cell lines in culture with different IC50 values [74 (compound 7), 98 (compound 5) and 180 microg/ml (compounds 1-4 and 6)].