Earthworm bioluminescence: characterization of high specific activity Diplocardia longa luciferase and the reaction it catalyzes

Diplocardia longa luciferase purified by an improved procedure differs from that first described by Bellisario et al. [Bellisario, R., Spencer, T. E., & Cormier, M. J. (1972) Biochemistry 11, 2256-2266] in having much higher specific activity (40X) and firmly bound, EPR-silent copper. Improved assay conditions suggest that this protein acts as a catalyst in a bioluminescent reaction involving the degradation of 3-(isovalerylamino)-1-hydroxypropane hydroperoxide. This substrate is formed spontaneously on the addition of hydrogen peroxide to D. longa luciferin (3-(isovalerylamino)propanal). The quantum yield of the bioluminescence for this substrate is 3%. Detailed physical and chemical analyses of high specific activity D. longa luciferase indicate that it is a large (300000 daltons), asymmetric (f/fo=1.63, with 0.4 g/g hydration), multisubunit enzyme. It contains carbohydrate (6%), lipid (2%), and copper (up to 4 mol/30000 daltons). The amino acid composition is unusual with 11% by weight of the residues being either proline or hydroxyproline.     

beta-Furfuryl-beta-glucoside. An endogenous activator of higher plant UDP-glucose: (1----3)-beta-glucan synthase.

We have recently established the existence of endogenous activators of higher plant UDP-glucose: (1----3)-beta-glucan synthase (Callaghan, T., Ross, P., Weinberger-Ohana, P., and Benziman, M. (1988) Plant Physiol. 86, 1099-1103). Here we report the purification and chemical analysis of the most abundant and specific compound, termed Activator I, isolated from Vigna radiata. This compound was extensively purified by a multistep procedure which yielded 0.1 mg of purified activator/g of fresh tissue. Enzyme digestion, neutral sugar analysis, GC/MS of permethylated derivatives, and NMR analysis of native Activator I indicated that the compound contains a single beta-linked glucosyl residue. High resolution FAB-MS indicated an elemental composition of C11H16O7 (Mr = 260), with a calculated Mr of 98 for the aglycone. 13C, DEPT, and COSY NMR spectra showed that the aglycone molecule is an oxygen heterocycle of 5 carbons, consistent with a structure of beta-furfuryl alcohol. Comparison of IR and GC/EI-MS spectra of authentic beta-furfuryl alcohol with native aglycone confirmed the conclusion that Activator I is beta-furfuryl-beta-glucoside. Chemically synthesized beta-furfuryl-beta-glucoside has identical chemical properties and biological activity when compared with the purified endogenous activator (Ka = 50 microM).     

A bifunctional luminogenic substrate for two luminescent enzymes: firefly luciferase and horseradish peroxidase.

Horseradish peroxidase (HRP) catalyzes the oxidative chemiluminescent reaction of luminol, and firefly luciferase catalyzes the oxidation of firefly D-luciferin. Here we report a novel substrate, 5-(5'-azoluciferinyl)-2,3-dihydro-1,4-phthalazinedione (ALPDO), that can trigger the activity of HRP and firefly luciferase in solution because it contains both luminol and luciferin functionalities. It is synthesized by diazotization of luminol and its subsequent azo coupling with firefly luciferin. NMR spectral data show that the C5' of benzothiazole in luciferin connects the diazophthalahydrazide. The electronic absorption and fluorescence properties of ALPDO are different from those of its precursor molecules. The chemiluminescence emission spectra of the conjugate substrate display biphotonic emission characteristic of azophthalatedianion and oxyluciferin. It has an optimum pH of 8.0 for maximum activity with respect to HRP as well as luciferase. At pH 8.0 the bifunctional substrate has 12 times the activity of luminol but has 7 times less activity than the firefly luciferin-luciferase system. The specific enhancement of light emission from the cyclic hydrazide part of ALPDO helped in the sensitive assay of HRP down to 2.0 x 10(-13) M and of ATP to 1.0 x 10(-14) mol. Addition of enhancers such as firefly luciferin and p-iodophenol (PIP) to the HRP-ALPDO-H2O2 system enhanced the light output.     

Role of a luciferin-binding protein in the circadian bioluminescent reaction of Gonyaulax polyedra

A luciferin-binding protein (LBP), which binds and protects from autoxidation the substrate of the circadian bioluminescent reaction of Gonyaulax polyedra, has been purified to near homogeneity. The purified protein is a dimer with two identical 72-kDa subunits, and an isoelectric point of 6.7. LBP is a major component of the cells, comprising about 1% of the total protein during the night phase, but drops to only about 0.1% during the day. The luciferin is protected from autoxidation by binding to LBP, and one luciferin is bound per dimer at alkaline pH (Ka approximately 5 x 10(7) M-1). The protein undergoes a conformational change with release of luciferin at pH values below 7, concurrent with an activation of Gonyaulax luciferase. LBP thus has a dual role in the circadian bioluminescent system.     

Structural Determination of a Repellent Principle in the Essential Oil of Mentha haplocalyx Briq.

Mentha haplocalyx Briq. is a plant which belongs to the Mentha genus of the Labiatae family. Yellowish-green oil is obtained from the leaves and stems with 0.05% yield by steam-distillation. Then the oil is fractionated and the isolated compound is purified by recrystallization. White prism crystal is obtained. It is identified as d-8-acetoxycarvotanacetone by UV, IR, NMR and MS. The mixed melting point of synthetic and natural samples has no depression. Their physical constants and spectral behavior also agree with each other. Thus, the structure of the crystalline compound isolated from the oil of Men, ha haplocalyx Briq. is confirmed to be d-8-acetoxycarvotanacetone. The compound has good repellent effect to mosquitoes, gnats and gadflies.     

菜油基长链辣椒素酯小型化合物库的制备与初步生物活性

以脂肪酶在有机介质中催化食用菜油与香草醇直接转酯反应合成了一个含菜油酰基的长链辣椒素酯小型化合物库。反应条件：5mmol香草醇,2．5mmol食用菜油溶于100mL丙酮中,加入0．5g固定化脂肪酶（Nov0435）,置于30℃的摇床上以200r／min反应24h。分离固定化酶后,反应物以硅胶柱色谱分离,得到长链辣椒素酯小型库,收率34．1％。这个化合物库主要由亚麻酸香草醇酯（1）、亚油酸香草醇酯（2）、软脂酸香草醇酯（3）、油酸香草醇酯（4）、硬脂酸香草醇酯（5）、花生-烯酸香草醇酯（6）、芥酸香草醇酯（7）组成;它在50彬mL时对PPARv模型具有弱的激活作用,激活倍数为1．5。用制备性高效液相色谱对这个化合物库进行分离,得到化合物1—4、7,以1HNMR确证了结构,其中化合物7未见文献报道。     

Enzymatic synthesis of palm-based ascorbyl esters

The synthesis of palm-based ascorbyl esters through transesterification of ascorbic acid and palm oil in tert-amyl alcohol catalyzed by immobilized lipase is described. Highest conversion (70–75%) was determined after 16 h reaction at 40 °C using lipase (Novozyme 435 from Candida antartica) with an ascorbic acid to palm oil mole ratio of 1:8. The purified product was further characterized by ¹³C NMR and GC–MS and the mixture of ascorbyl monoesters obtained were identified as ascorbyl monooleate (61%), ascorbyl monopalmitate (30%) and ascorbyl monostearate (9%). The antioxidant activity of palm-based ascorbyl esters was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) test. The results showed that pure palm-based ascorbyl esters have an antioxidant activity with an IC₅₀ value of 0.1 mg/mL.     

Studies in bioluminescence IV. Properties of luciferin from Pholas dactylus

Light could be obtained by the addition of Fe²⁺ to purified luciferin from Pholas dactylus in the absence of luciferase. The total light emitted was proportional to the concentration of luciferin used. The characteristics of this nonenzymic emission correspond to those of the fast reaction previously described. It may have a physiological importance since iron is present in the luciferin. The injection of Fe²⁺ alone was not sufficient; the presence of a complexing agent such as phosphate or CN⁻ or EDTA was also necessary. Light emission could also be obtained by the addition of H₂O₂, in the presence of Fe²⁺, to luciferin. It has been demonstrated that, for a given amount of luciferin, the total light emitted by the action of varying ratios of Fe²⁺ and luciferase is constant.     

Phytochemical investigation and evaluation of anti-inflammatory and anti-arthritic activities of essential oil of Strobilanthus ixiocephala Benth

Column chromatographic fractionation of essential oil obtained by hydrodistillation from the flowering tops of S. ixiocephala resulted in the isolation of beta-caryophyllene, fenchyl acetate, T-cadinol and a new sesquiterpene alcohol for which a name ixiocephol has been proposed. The beta-caryophyllene and fenchyl acetate were identified by Co-TLC with authentic samples whereas T-cadinol and ixiocephol were structurally elucidated by UV, IR, 1H NMR, 13C NMR and Mass spectral data. The GC-MS analysis of the essential oil has also revealed the presence of various monoterpenoids and sesquiterpenoids. The essential oil of S. ixiocephala demonstrated a dose dependant anti-inflammatory activity in carrageenan-induced rat paw oedema. It has also revealed good activity in cotton pellet granuloma and adjuvant induced arthritis model in rats.     

Isolation and identification of the 3-hydroxy-5-hydroxymethyl-pyridinium compound as a novel advanced glycation end product on glyceraldehyde-related Maillard reaction

Glyceraldehyde (200 mM) and N alpha-acetyllysine (100 mM) were incubated in 0.2 M sodium phosphate buffer (pH 7.4) at 37 degrees C for a week. A major compound, glyceraldehyde-related Maillard reaction product, was purified from the reaction mixture using reverse phase (ODS)-HPLC. It was identified as 1-(5-acetylamino-5-carboxypentyl)-3-hydroxy-5-hydroxymethyl-pyridinium, named as GLAP (Glyceraldehyde derived Pyridinium compound), using NMR and MS analyses. It was suggested that GLAP as a novel advanced glycation end product (AGE) is one of the key compounds in the glyceraldehyde-related Maillard reaction.     

In vitro metabolism of [2-13C]-ethanol by 1H NMR spectroscopy using 13C decoupling with the reverse dept polarization-transfer pulse sequence

The metabolism of [2-13C]-ethanol by alcohol dehydrogenase purified from Drosophila melanogaster has been observed by proton nuclear magnetic resonance spectroscopy (NMR). The reverse-DEPT pulse sequence, with composite pulse 13C decoupling to simplify and increase the signal-to-noise of spectra, has been used to eliminate the strong water signal while still observing the proton signals of metabolites of interest. Using these techniques the rates of synthesis of acetaldehyde, its diol and acetate from [2-13C] ethanol by alcohol dehydrogenase were measured simultaneously.     

Purification and properties of Renilla reniformis luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis (Renilla luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5.) catalyzes the bioluminescent oxidation of Renilla luciferin producing light (lambdaB 480 nm, QB 5.5%), oxyluciferin, and CO2 (Hori, K., Wampler, J.E., Matthews, J.C., and Cormier, M.J. (1973), Biochemistry 12, 4463). Using a combination of ion-exchange, molecular-sieve, sulfhydryl-exchange, and affinity chromatography, luciferase has been purified, approximately 12 000-fold with 24% recovery, to homogeneity as judged by analysis with disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and ultracentrifugation. Renilla luciferase is active as a nearly spherical single polypeptide chain monomer of 3.5 X 10(4) daltons having a specific activity of 1.8 X 10(15) hp s-1 mg-1 and a turnover number of 111 mumol min-1 mumol-1 of enzyme. This enzyme has a high content of aromatic and hydrophobic amino acids such that it has an epsilon280nm 0.1% of 2.1 and an average hydrophobicity of 1200 cal residue-1. The high average hydrophobicity of luciferase, which places it among the more hydrophobic proteins reported, is believed to account, at least in part, for its tendency to self-associate forming inactive dimers and higher molecular weight species.     

Oplophorus oxyluciferin and a model luciferin compound biologically active with Oplophorus luciferase.

The luciferin of the bioluminescent decapod shrimp, Oplophorus gracilorostris, was purified and studied with respect to u.v. spectrum, fluorescence spectrum, mass spectrum and luminescent cross-reaction with the enzyme luciferase of the bioluminescent ostracod, Cypridina hilgendorfii. On the basis of these results, an empirical formula C10H13N3O3 and an imidazo [1,2-a]pyrazin-3-one structure are proposed for luciferin. Of three model luciferin compounds, 3-hydroxy-2-methylimidazo[1,2-a]pyridine is biologically active with both Oplophorus and Cypridina luciferase, indicating that a pyrazine structure is not essential for biological activity with Cypridina luciferase.     

4-hydroxy-2-nonylquinoline: a novel iron chelator isolated from a bacterial cell membrane

The membrane associated iron chelator of Pseudomonas aeruginosa has been extracted from membranes of iron-rich cells with ethanol and purified by reverse phase HPLC. Using 13C NMR and FAB mass spectroscopy, the structure of the chelator has been determined to be 4-hydroxy-2-nonylquinoline. This compound has been previously isolated and named pseudan IX, a name which we use here. We synthesized pseudan IX and show that the spectral properties of the synthesized compound and the purified compound are nearly identical. Also purified from the ethanol extract of membranes is 4-hydroxy-2-heptylquinoline, i.e., pseudan VII. Bacterially purified pseudan IX binds iron as indicated by the incorporation of radiolabeled iron into the chelator and by the formation of pink micelles in a concentrated ethanol extract. The formation of pink micelles upon addition of iron to the synthesized compound indicates that it binds iron.     

Dinoflagellate luminescence: purification of a NAD(P)H-dependent reductase and of its substrate

The soluble enzymatic luminescent system of the dinoflagellate Pyrocystis lunula (luciferase-luciferin) is coupled with an enzymatic NAD(P)H-dependent reaction. The enzyme is a soluble reductase (Mr 47,000) which catalyzes, in the presence of NAD(P)H, the reduction of a molecule called P630. Reduced P630 has the same spectral characteristics as the purified luciferin. The luciferase can oxidize this reduced molecule with a light emission at 480 nm. These observations suggest that reduced P630 is a luciferin molecule. The oxidized form seems, in these conditions, to be the precursor of luciferin.     

Bioactive compounds of the volatile oil of Dracocephalum kotschyi

Trypanocidal activity was found in the volatile oil of dried Dracocephalum kotschyi. GC-MS analysis determined that the major constituents of the oil were geranial (35.8%), C10H14O (26.6%), limonene (15.8%) and 1,1-dimethoxy decane (14.5%). In order to isolate the unknown biologically active monoterpene, fractionation of the volatile oil was carried out by silica gel column chromatography. The structure of the oxygenated compound was confirmed to be limonene-10-al (C10H14O) by analysis of physical and spectroscopic data (1H NMR, 13C NMR, HMBC and HMQC).     

STUDIES ON BIOLUMINESCENCE : IX. CHEMICAL NATURE OF CYPRIDINA LUCIFERIN AND CYPRIDINA LUCIFERASE.

There seems to be very little doubt but that luciferase is a protein or so closely associated with proteins that their removal destroys its characteristic properties. The particular group of proteins to which it belongs may be arrived at by a process of exclusion, and only the group of albumins has properties which agree completely with those of luciferase. Dubois believes Pholas luciferase to be an oxidizing enzyme similar to the oxydones of Battelli and Stern because it is readily destroyed by fat solvents such as chloroform, strong alcohol, etc. He has detected iron in a luciferase solution which has dialyzed against running water for a long time, and believes it to be made up of protein in combination with iron and to act as an "oxyzymase ferrique." Cypridina luciferase, on the other hand, is not readily destroyed by fat solvents. Toluene and chloroform are good preservatives, and I often make use of them for this purpose, keeping the luciferase solutions for many months. Professor A. H. Phillips of Princeton University has very kindly analyzed some whole dried Cypridinœ for me and finds iron, copper, and manganese but no zinc or vanadium to be present. Whether these metals are connected with the action of Cypridina luciferase is uncertain, but it is significant that all three of the metals thought to be concerned in organic oxidations are present. Although a large amount of luciferin mixed with a small amount of luciferase will use up all the latter, I agree with Dubois that luciferase has sufficient properties in common with the enzymes as a class to be considered an enzyme. The peroxidases are well known to be used up in the reactions they accelerate. All workers on enzymes agree that the more enzymes are purified the less active they become. The chemical procedures necessary to remove foreign material bring about irreversible changes in the enzyme itself, a characteristic also of many protein groups and of the colloidal state in general. This is true in the case of luciferase, for the crude luciferase solution is the most active preparation that can be obtained. I believe that Cypridina luciferase should be placed in a class of oxidizing enzymes by itself—a group having the chemical reactions of an albumin, possibly in combination with some heavy metal, and which as far as we know, acts specifically on only one substance, Cypridina luciferin. It resembles the plant peroxidases in resisting the action of chloroform, toluene, etc., but will not oxidize any of the hydroxyphenol or aminophenol compounds so readily oxidized by the peroxidases, nor will the peroxidases oxidize luciferin with light production. Dubois'' researches show that Pholas luciferase differs in some properties from Cypridina luciferase, and my own work indicates that firefly luciferase is more like that of Pholas. A comparative study of other species of luminous animals is needed in order to delimit more accurately the class of luciferases as a whole. Luciferin presents many characteristics in common with the proteins, but two, which, to say the least, throw doubt on its protein nature: (1) its peculiar solubility (in alcohols, esters, and glacial acetic acid), (2) and its resistance to digestion by proteases, even by trypsin which has almost universal digestive action. These two peculiarities have been discussed above. We can only say that if a protein, luciferin must belong to a new group differing from known natural proteins in these respects. In general characteristics this new group would fall somewhere on the border-line between the proteoses and peptones. It would not be surprising to find in nature proteoses or peptones soluble in absolute alcohol. We know also that some NH-CO linkages of proteins are broken down with great difficulty by trypsin as it is difficult to obtain a tryptic digest of protein which does not give the biuret reaction, and the work of Fischer and Abderhalden has shown that certain artificial polypeptides are not digested by pure activated pancreatic juice. We have, then, three possibilities. Luciferin is (1) either a natural proteose not attacked by trypsin, or (2) if attacked by trypsin, its decomposition products (presumably amino-acids) still contain the group oxidizable with light production, or (3) it is not a protein at all. I believe that luciferin has too many protein characteristics to conform to the last possibility. I have been unable to oxidize with light production various mixtures of amino-acids (from beef and casein) by means of luciferase and consequently am led to believe that luciferin is a new natural proteose, soluble in absolute alcohol and not digested by trypsin. Dubois believes Pholas luciferin to be a natural albumin with acid properties. Cypridina luciferin could not possibly be regarded as an albumin, but it is very likely that the luciferins of different species of luminous animals differ in certain characteristics. As in the case of the luciferases, we know that the luciferins are not identical substances, and only future work can determine in what particulars they differ. A summary of the properties of Cypridina luciferin and Cypridina luciferase will be found in the tables accompanying this paper.     

Moringa oleifera oil: a possible source of biodiesel

Biodiesel is an alternative to petroleum-based conventional diesel fuel and is defined as the mono-alkyl esters of vegetable oils and animal fats. Biodiesel has been prepared from numerous vegetable oils, such as canola (rapeseed), cottonseed, palm, peanut, soybean and sunflower oils as well as a variety of less common oils. In this work, Moringa oleifera oil is evaluated for the first time as potential feedstock for biodiesel. After acid pre-treatment to reduce the acid value of the M. oleifera oil, biodiesel was obtained by a standard transesterification procedure with methanol and an alkali catalyst at 60 degrees C and alcohol/oil ratio of 6:1. M. oleifera oil has a high content of oleic acid (>70%) with saturated fatty acids comprising most of the remaining fatty acid profile. As a result, the methyl esters (biodiesel) obtained from this oil exhibit a high cetane number of approximately 67, one of the highest found for a biodiesel fuel. Other fuel properties of biodiesel derived from M. oleifera such as cloud point, kinematic viscosity and oxidative stability were also determined and are discussed in light of biodiesel standards such as ASTM D6751 and EN 14214. The (1)H NMR spectrum of M. oleifera methyl esters is reported. Overall, M. oleifera oil appears to be an acceptable feedstock for biodiesel.     

Ascorbic acid conjugates isolated from the phloem of Cucurbitaceae

Analysis of phloem exudates from the fruit of Cucurbitaceae revealed the presence of several compounds with UV-visible absorption spectra identical to that of l-ascorbic acid. In Cucurbita pepo L. (zucchini), the compounds could be isolated from phloem exudates collected from aerial parts of the plant but were not detected in whole tissue homogenates. The compounds isolated from the phloem exudates of C. pepo fruit were eluted from strong anion exchange resin in the same fraction as l-ascorbic acid and were oxidised by ascorbate oxidase (E.C. 1.10.3.3). The major compound purified from C. pepo fruit exudates demonstrated similar redox properties to l-ascorbic acid and synthetic 6-O-glucosyl-l-ascorbic acid (6-GlcAsA) but differed from those of 2-O-glucosyl-l-ascorbic acid (2-GlcAsA) isolated from the fruit of Lycium barbarum L. Parent and fragment ion masses of the compound were consistent with hexosyl-ascorbate in which the hexose moiety was attached to C5 or C6 of AsA. Acid hydrolysis of the major C. pepo compound resulted in the formation of l-ascorbic acid and glucose. The purified compound yielded a proton NMR spectrum that was almost identical to that of synthetic 6-GlcAsA. A series of l-ascorbic acid conjugates have, therefore, been identified in the phloem of Cucurbitaceae and the most abundant conjugate has been identified as 6-GlcAsA. The potential role of such conjugates in the long-distance transport of l-ascorbic acid is discussed.     

Characterization of new biosurfactant produced by Klebsiella sp. Y6-1 isolated from waste soybean oil

To obtain predominant bacteria degrading crude oil, we isolated some bacteria from waste soybean oil. Isolated bacterial strain had a marked tributyrin (C4:0) degrading activity as developed clear zone around the colony after incubation for 24h at 37 degrees C. It was identified as Klebsiella sp. Y6-1 by analysis of 16S rRNA gene. Crude biosurfactant was extracted from the culture supernatant of Klebsiella sp. Y6-1 by organic solvent (methanol:chloroform:1-butanol) after vacuum freeze drying and the extracted biosurfactant was purified by silica gel column chromatography. When the purified biosurfactant dropped, it formed degrading zone on crude oil plate. When a constituent element of the purified biosurfactant was analyzed by TLC and SDS-PAGE, it was composed of peptides and lipid. The emulsification activity and stability of biosurfactant was measured by using hydrocarbons and crude oil. The emulsification activity and stability of the biosurfactant showed better than the chemically synthesized surfactant. It reduced the surface tension of water from 72 to 32 mN/m at a concentration of 40 mg/l.