Isolation and characterization of a novel haemoprotein b-559 from Bengal gram (Cicer arietinum).

A haemoprotein was purified to apparent homogeneity from Bengal-gram seeds. The purified protein exhibited an absorption maximum at 412 nm (Soret band) that upon reduction with dithionite gave rise to a shift in the Soret band to 426 nm with concomitant appearance of an alpha-band at 559 nm and a beta-band at 530 nm. In the reduced state the Bengal-gram haemoprotein showed reactivity towards CO, nitrite and hydroxylamine. SDS/polyacrylamide-slab-gel electrophoresis showed that the haemoprotein has Mr 78,000. Gel-filtration and ultracentrifugal analyses suggest that the Bengal-gram haemoprotein is oligomeric in nature. Since it differs from photosynthetic membrane cytochrome b-559 in solubility in buffer, in reactivity towards CO and in molecular size, it appears to be a novel haemoprotein b-559.     

Effect of an endogenous factor on arginase activity in germinating Bengal gram (Cicer arietinum) seeds

Arginase (EC 3.5.3.1) activity increased in the cotyledon, while it declined rapidly in the embryonic axis of Bengal gram ( Cicer arietinum L. cv. Desi type) seeds during germination. The decrease in enzyme activity in the embryonic axis was accompanied by changes in the properties of arginase in vitro such as decreased stability, increased heat lability and failure to bind to DEAE-cellulose. These alterations were due to the presence of a low-molecular-weight factor in the extract, which was purified and identified as a hexose derivative. During germination the concentration of the factor increased in the embryonic axis, while no detectable level of the factor was present in the cotyledons. We postulate that the factor may have a role in the regulation of arginase activity in vivo.     

Effect of nitrogen,phosphorus and potassium deficiency on the uptake and mobilization of ions in Bengal gram (Cicer arietinum)

Nitrogen, phosphorus and potassium deficiency reduced the uptake of³²P-phosphate,³⁵S-sulphate,²⁴Na-,⁴²K-,⁴⁵Ca-,⁵⁴Mn-,⁵⁹Fe- and⁶⁵Zn- byCicer arietinum (Bengal gram) cv B-75. Root length, leaf area and dry weight of the tissues were also reduced. Since in several cases, the total contents of the radio nucleides both on per plant and per unit dry weight basis were curtailed, the decrease in uptake of several ions cannot be entirely due to reduced growth rate. The reduction in³²P-phosphate uptake was more severe with nitrogen deficient plants than that in phosphate deficient ones; potassium deficient plants, however, took up⁴²K- as avidly as the control plants. Simultaneously the uptake of³⁵SO^{4 2}- and other cations was affected particularly by nitrogen deficiency. The distribution of radionucleides between the root and shoot portions was also disturbed in several cases by deficiency conditions. The radionucleides taken up accumulated in the young regions as in the case of pea and other dicotyledonous plants. Mobilization of³²P and³⁵S in the reproductive plants was most markedly affected by nitrogen and potassium-deficiency.     

The use of mustard (Brassica juncea L.) and gram (Cicer arietinum L.) seedling germination inhibition assay for aflatoxin B1

Seed germination and seedling growth as well as chlorophyll and carotenoid contents of mustard and gram seeds were inhibited or reduced significantly due to the treatment of different concentrations (100, 250, 500, 1000 and 2000 ng/ml) of aflatoxin B₁. The range of inhibition in all the parameters was found to be directly influenced by the concentrations of toxin applied. Mustard and gram seedling germination inhibition assay can be used for aflatoxin B₁.     

Removal of Acid Blue25 from aqueous solutions using Bengal gram fruit shell (BGFS) biomass

The feasibility for the removal of Acid Blue25 (AB25) by Bengal gram fruit shell (BGFS), an agricultural by-product, has been investigated as an alternative for high-cost adsorbents. The impact of various experimental parameters such as dose, different dye concentration, solution pH, and temperature on the removal of Acid Blue25 (AB25) has been studied under the batch mode of operation. pH is a significant impact on the sorption of AB25 onto BGFS. The maximum removal of AB25 was achieved at a pH of 2 (83.84%). The optimum dose of biosorbent was selected as 200 mg for the removal of AB25 onto BGFS. Kinetic studies reveal that equilibrium reached within 180 minutes. Biosorption kinetics has been described by Lagergren equation and biosorption isotherms by classical Langmuir and Freundlich models. Equilibrium data were found to fit well with the Langmuir and Freundlich models, and the maximum monolayer biosorption capacity was 29.41 mg g^?1 of AB25 onto BGFS. The kinetic studies indicated that the pseudo-second-order (PSO) model fitted the experimental data well. In addition, thermodynamic parameters have been calculated. The biosorption process was spontaneous and exothermic in nature with negative values of ΔG° (?1.6031 to ?0.1089 kJ mol^?1) and ΔH° (?16.7920 kJ mol^?1). The negative ΔG° indicates the feasibility of physical biosorption process. The results indicate that BGFS could be used as an eco-friendly and cost-effective biosorbent for the removal of AB25 from aqueous solution.     

An acid stable trypsin-chymotrypsin inhibitor from horse gram (Dolichos biflorus)

An inhibitor of trypsin and chymotrypsin was purified from horse gram (Dolichos biflorus) beans. The concentration of the inhibitor which provided total inhibition was 0.27 Μg/Μg tryptic enzyme and 0.46 Μg/Μg chymotryptic enzyme. The inhibitor was stable at 37‡C between pH of 3 to 11 and at 97‡C, upto pH 5.0 only. While the activities were rapidly lost in 0.1N NaO H the loss was only 5 0% in 0.1N HCl when kept for 2 h at 97‡C. On heating at pH 7.8, it remained stable upto 80‡C with a gradual loss in activities at 97‡C and a total loss occurring by autoclaving at 15 psi for 10 min. Reduction of disulphide bonds by 2-mercapto-ethanol, pronase digestion and boiling in the presence of 1 M NaCl led to reduction in the activities. However, the inhibitor was resistant to the action of pepsin and subtilisin and to urea at 37‡C.     

N-dealkylation of aminopyrine catalyzed by soybean lipoxygenase in the presence of hydrogen peroxide

A hypothesis that lipoxygenase may mediate N-dealkylation of xenobiotics was investigated using the prototype drug aminopyrine and soybean lipoxygenase as a model enzyme in the presence of hydrogen peroxide. Formaldehyde production as a result of N-demethylation of aminopyrine exhibited pH optimum of 6.5. The reaction was dependent on the incubation time, amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. Under the experimental conditions employed, the specific activity for N-demethylation of aminopyrine was found to be 823 ± 93 nmoles per min/mg protein or 89 ± 10 nmoles per min/nmole of enzyme. The reaction was significantly inhibited by nordihydroguaiaretic acid and gossypol, the classical inhibitors of lipoxygenase. Spectrophotometric analyses indicated the generation of a nitrogen-centered free-radical cation as the initial oxidation product of aminopyrine. The rate of accumulation of this radical species was also dependent on pH, the amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. The radical production was markedly suppressed by ascorbate, glutathione, and dithiothreitol in a concentration-dependent manner. Preliminary data gathered for the oxidation of other chemicals indicated that the lipoxygenase exhibits a unique substrate specificity. Collectively, the evidence presented suggests for the first time that lipoxygenase pathway may be involved in N-demethylation of aminopyrine and other chemicals. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 175–183, 1998     

Effect of Feeding Bengal Gram (Cicer arietinum) at Different Protein Levels on Plasma Lipids of Rats

L-Amino acid ligase catalyzes the formation of an α-peptide bond from unprotected L-amino acids in an ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin. Rhizocticins are dipeptide or tripeptide antibiotics and commonly possess L-arginyl-L-2-amino-5-phosphono-3-cis-pentenoic acid. The purification was carried out by detecting L-arginine hydroxamate synthesis activity, and a target enzyme was finally purified 1,280-fold with 0.8% yield. The corresponding gene was then cloned and designated rizA. rizA was 1,242 bp and coded for 413 amino acid residues. Recombinant RizA was prepared, and it was found that the recombinant RizA synthesized dipeptides whose N-terminus was L-arginine in an ATP-dependent manner. RizA had strict substrate specificity toward L-arginine as the N-terminal substrate; on the other hand, the substrate specificity at the C-terminus was relaxed.     

The binding of copper ions to glycine-rich proteins (GRPs) from Cicer arietinum

Cicer arietinum GRP1 and GRP2 are rich in glycine interposed with histidine and tyrosine. In order to study whether or not these proteins bind Cu(2+), circular dichroism (CD) and nuclear magnetic resonance (NMR) were measured for three synthetic peptides corresponding to sections of the protein's sequences including 1, N(1)Y(2)G(3)H(4)G(5)G(6)G(7)N(8)Y(9)G(10)N(11), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. The visible CD spectra for 1 showed a positive peak near 590 nm not at pH 6.0 but pH 7.4 in the presence of copper ions. The Cu(2+) binding induced a drastic change in the far-UV CD spectra, showing the occurrence of large conformation changes. In the 2D TOCSY NMR spectra at pH 7.4, the addition of small amounts of CuSO(4) caused a significant broadening of proton resonances of not only His4 but also Gly5, Asn8 and Asn11. CD titration experiment suggested that NYGHGGGNYGN including one repeat unit comprises the fundamental Cu(2+) binding unit.     

Stereocontrol of arachidonic acid oxygenation by vertebrate lipoxygenases: newly cloned zebrafish lipoxygenase 1 does not follow the Ala-versus-Gly concept

Animal lipoxygenases (LOXs) are classified according to their specificity of arachidonic acid oxygenation, and previous sequence alignments suggested that S-LOXs contain a conserved Ala at a critical position at the active site but R-LOXs carry a Gly instead. Here we cloned, expressed, and characterized a novel LOX isoform from the model vertebrate Danio rerio (zebrafish) that carries a Gly at this critical position, classifying this enzyme as putative arachidonic acid R-LOX. Surprisingly, the almost exclusive arachidonic acid oxygenation product was 12S-H(p)ETE (hydro(pero)xyeicosatetraenoic acid), and extensive mutation around Gly-410 failed to induce R-lipoxygenation. This finding prompted us to explore the importance of the corresponding amino acids in other vertebrate S-LOXs. We found that Ala-to-Gly exchange in human 15-LOX2 and human platelet 12-LOX induced major alterations in the reaction specificity with an increase of specific R-oxygenation products. For mouse 5-LOX and 12/15-LOX from rabbits, men, rhesus monkeys, orangutans, and mice, only minor alterations in the reaction specificity were observed. For these enzymes, S-HETE (hydroxyeicosatetraenoic acid) isomers remained the major oxygenation products, whereas chiral R-HETEs contributed only 10-30% to the total product mixture. Taken together these data indicate that the Ala-versus-Gly concept may not always predict the reaction specificity of vertebrate LOX isoforms.     

Seasonal variation in hydrochloric acid, malic acid, and calciumions secreted by the trichomes of chickpea (Cicer arietinum)

A novel experimental set-up and method of recording of electrical potential differences in plants have been developed which enable continuous, 8-channel monitoring of electrical activity over extended periods of time using inserted, extracellular electrodes. The investigations were carried out on 21- to 23-day-old Helianthus annuus plants, and spontaneously-generated action potentials were recorded during monitoring sessions lasting for 3 days and nights. Characteristics of these spontaneous action potentials were elaborated, adopting as parameters their typical form, amplitude, duration, velocity, direction, and distance of propagation and frequency of occurrence in morphologically different parts of the plant, Variability, similarities, and interdependence of the above parameters in individual plants and in a group of 15 plants were determined. A hypothesis concerning propagation of action potentials in plants along specific impulse-propagating 'columns' is discussed. The frequency of generated impulses is highest at night and lowest in the day and also displays an apparent 24-h rhythm. Presumably this mechanism is under both endogenous and exogenous control and may be partly dependent on a biological clock.     

Hydration of linoleic acid by bacteria isolated from ruminants

Two strains of Enterococcus faecalis isolated from the ovine rumen and known to hydrate oleic acid were shown to transform linoleic acid by hydration into two products. The products, identified as 10-hydroxy-12-octadecenoic acid and 13-hydroxy-9-octadecenoic acid, were formed during stationary phase in yields of 13% and 6% respectively. Yields increased to 22% and 14% when culture conditions were optimised. To our knowledge, this is the first report of 13-hydroxy-9-octadecenoic acid production by bacteria. During a search for further linoleic-acid-hydrating bacteria, a strain of Streptococcus bovis isolated from bovine faeces and the ruminal strain S. bovis JB1 were found to hydrate linoleic acid. Both strains formed only one product and the most rapid appearance occurred during exponential growth. The S. bovis product, identified as 13-hydroxy-9-octadecenoic acid, formed in a yield of 28%. This study provides the first information on linoleic acid hydration by ruminal bacteria.     

Hydroperoxide isomers and ketohydroxy product from oxidation of linoleic acid by eggplant lipoxygenase

Hydroperoxides produced by oxidation of linoleic acid with purified eggplant lipoxygenase were separated by TLC and analysed by IR spectroscopy. The methyl hydroxystearates from the enzymatically produced hydroperoxides were analysed by MS and GLC. Both analyses indicated that the eggplant enzyme converted linoleic acid almost exclusively (96%) into the 13-hydroperoxy isomer whereas the 9-hydroperoxy isomer was only a minor product (4%). HPLC of the methyl ester of the isolated hydroperoxides showed three components. Each component was collected, reduced to methyl hydroxystearate and characterized by GLC, MS and IR analysis. The components were identified as 13-hydroperoxy cis-trans isomer (92.8%), 13-hydroperoxy trans-trans isomer (2.6%) and 9-hydroperoxy cis-trans isomer (4.6%). A polar by-product present in the reaction mixture was identified by IR, ¹H NMR, and MS (of the toluene-p-sulphonyl derivative) as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid.     

Effect of Aluminum on the Production of Siderophore by Rhizobium sp. (Cicer arietinum)

Rhizobium sp. strain BICC 651 in the presence of 100 μM Al³⁺ produced a threefold higher level of siderophore than in the control culture under iron limitation during the stationary phase. Al³⁺ in increasing concentrations resulted in decreased growth, and the effect was alleviated by the addition of iron. Siderophore production decreased gradually in Al³⁺-treated culture as well as in the control with the addition of increasing concentrations of Fe³⁺, and at 50 μM Fe³⁺ the level of siderophore was practically undetectable. The siderophore binds Fe³⁺ and also Al³⁺. The outer membrane protein profiles of the bacteria grown in the presence or absence of Al³⁺ were indistinguishable. Received: 15 November 1999 / Accepted: 21 December 1999     

Partial purification and some biochemical properties of acid phosphatase in germinating chickpea (Cicer arietinum) seeds

An acid phosphatase (EC 3.1.3.2.) from the embryonic axes of chickpea seeds ( Cicer arietinum L. cv. Castellana) was purified by ammonium sulphate precipitation, chromatography on Sephacryl S-200 and polyacrylamide gel electrophoresis. The preparation has an apparent molecular weight of 39 kDa, pH optimum for p -nitrophenylphosphate hydrolysis of 5.25, and K _m of 0.57 m M . The enzyme hydrolyzed all the mono- and di-phosphorylated sugars tested, but had no effect on ATP, ADP, AMP and phosphoenolpyruvate. Phosphate was a competitive inhibitor. Mg²⁺. Ca²⁺, Hg²⁺, Fe³⁺, arsenate, K⁺ and Zn²⁺ were inhibitory. Mn²⁺, dithiothreitol and EDTA had no effect, and polyamines were activators.     

Improvement of ethanol production in very high gravity fermentation by horse gram (Dolichos biflorus) flour supplementation

AIMS: To determine the effect of osmotic stress on yeast and to investigate the protective role of horse gram flour during very high gravity (VHG) ethanol fermentation. METHODS AND RESULTS: Saccharomyces cerevisiae was inoculated into high sugar (30-40%, w/v) containing medium with and without supplementation of horse gram flour. The fermentation experiments were carried out in batch mode. The effect of 4 or 6% of horse gram flour to the medium on the metabolic behaviour and viability of yeast was studied. Significant increase in ethanol yield up to 50% and dramatic decrease in glycerol production up to 100% was observed in the presence of horse gram flour. The fermentation rate was increased from 3 to 5 days with increased viable cell count. The physical and chemical factors of horse gram flour may aid in reducing the osmotic stress of high gravity fermentation of ethanol as well as enhancing ethanol yield. CONCLUSIONS: It was found that horse gram flour not only reduced fermentation time but also enhanced ethanol production by better utilization of sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of high ethanol concentration by using VHG sugar fermentation eliminates the expensive steps in the conventional process and saves time.     

On the mechanism of the oxygenation of arachidonic acid by human platelet lipoxygenase

Formation of 12L-hydroxy-5,8,10,14-eicosatetraenoic acid from [10L-³H; 3-¹⁴C]arachidonic acid in suspensions of human platelets occurred with extensive loss of tritium and was accompanied by an isotope effect. These experiments showed that there is an antarafacial relation between the elimination of hydrogen from C-10 and insertion of oxygen at C-12 by human platelet lipoxygenase, and that the hydrogen elimination probably occurs as the initial step of the conversion. (Endo) peroxide intermediates formed by the fatty acid cyclo-oxygenase pathway activated platelet lipoxygenase.