An immunological and enzymological survey of asparaginase in seeds of Lupinus

Rabbit antiserum was raised against potassium-independent asparaginase purified from Lupinus polyphyllus. A survey of 54 lines of Lupinus showed that only 11 contained the enzyme in maturing cotyledons with activities > 0.5 μmol/hr per g fr. wt. Potassium-dependent asparaginase activity was detected in a number of the remaining varieties.     

Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase

Asparaginase was purified from Helicobacter pylori 26695 and its pathophysiological role explored. The K(m) value of asparagine was 9.75 ± 1.81 μM at pH 7.0, and the optimum pH range was broad and around a neutral pH. H. pylori asparaginase converted extracellular asparagine to aspartate. H. pylori cells were unable to take up extracellular asparagine directly. Instead, aspartate produced by the action of the asparaginase was transported into H. pylori cells, where it was partially converted to β-alanine. Asparaginase exhibited striking cytotoxic activity against histiocytic lymphoma cell line U937 cells via asparagine deprivation. The cytotoxic activity of live H. pylori cells against U937 cells was significantly diminished by deletion of the asparaginase gene, indicating that asparaginase functions as a cytotoxic agent of the bacterium. The cytotoxic effect was negligible for gastric epithelial cell line AGS cells, suggesting that the effect differs across host cell types. An asparaginase-deficient mutant strain was significantly less capable of colonizing Mongolian gerbils. Since asparagine depletion by exogenous asparaginase has been shown to suppress lymphocyte proliferation in vivo, the present results suggest that H. pylori asparaginase may be involved in inhibition of normal lymphocyte function at the gastric niche, allowing H. pylori to evade the host immune system.     

Some properties of glutamine synthetase and glutamate synthase from Derxia gummosa

The incorporation of ¹⁵N into washed cells of Derxia gummosa from labelled-(NH₄)₂SO₄ and -KNO₃ respectively was inhibited by both L-methionine-DL-sulphoximine and azaserine. Glutamine synthetase purified to homogeneity from this bacterium had a molecular weight of 708 000 and was composed of 12 similar subunits each of 59 000. The enzyme assayed by γ-glutamyltransferase method had K_m values for L-glutamine and hydroxylamine of 12.5 and 1.2 mM, respectively. Optimal pH values for adenylylated and deadenylylated forms were pH 7.0 and pH 8.0, respectively. The adenylylated enzyme was deadenylylated by treatment with snake venom phosphodiesterase. The inhibitions by both glutamate and ammonia were competitive. The activity was markedly inhibited by L-methionine-DL-sulphoximine, alanine, glycine and serine and to a lesser extent by aspartate, phenylalanine and lysine. Various tri-, di- and mono-phosphate nucleotides, organic acids (pyruvate, oxalate and oxaloacetate) were also inhibitory. Glutamate synthase purified 167-fold had specific requirements for NADH, L-glutamine and 2-ketoglutarate. The K_m values for NADH, glutamine and 2-ketoglutarate were 9.6, 270 and 24 μM respectively. Optimal pH range was 7.2–8.2. The enzyme was inhibited by azaserine, methionine, aspartate, AMP, ADP and ATP.     

Alkaloid distribution in some new world Lupinus species

Alkaloidal profiles of 21 Lupinus species indigenous to North and South America have been determined. Nineteen quinolizidine alkaloids were identified, including aphyllidine and N-methylcytisine, which have not previously been found in the genus. Two dipiperidine alkaloids were also detected. The pattern of alkaloidal distribution is related to a taxonomic classification of the genus.     

Enzymes of ammonia assimilation in root nodules of Trigonella foenum-graecum

The main pathway of ammonia assimilation in the root nodules of Trigonella foenum-graecum is via nodule cytosol glutamine synthetase-glutamate synthase.     

Regulation of the assimilation of nitrate in Chlamydomonas reinhardii

In Chlamydomonas, The assimilation of ammonia proceeds through the glutamine synthetaseglutamate synthase pathway. The primary target in the regula     

Glutamine-dependent asparagine synthetase from Lupinus luteus

Asparagine synthetase (glutamine-hydrolyzing [l-aspartate: l-glutamine amido-ligase (AMP-forming), E.C. 6.3.5.4] was purified over 500-fold from cotyledon extracts of 1-week-old yellow lupin seedlings. The enzyme was labile and required protection by high levels of thiols; glycerol and the substrates also stabilized it. The reaction products were shown to be asparagine, AMP, PPi and glutamate. The limiting K_m values were for aspartate 1·3 mM, for MgATP 0·14 mM and for glutamine 0·16 mM. Positive homotropic cooperativity was observed for MgATP only, and gel filtration studies indicated that the substrate-free enzyme (MW 160 000) associated to a dimer (MW 320 000 in the presence of MgCl₂ and ATP. The purified enzyme, which had some glutaminase activity, catalyzed an aspartate- and glutamine-independent ATP-PPi exchange reaction at a rate 5–7-fold higher than the rate of asparagine synthesis. Initial velocity studies and exchange data indicated an overall ping-pong mechanism. Compared to similar enzymes isolated from mammalian tumor cells, the lupin enzyme appears to be unique with respect to MW, reaction mechanism and regulatory properties. The allosteric properties observed suggest an important role for this enzyme in the regulation of asparagine biosynthesis.     

Peroxidase from euphorbia characias latex: purification and properties

A peroxidase has been purified to homogeneity from Euphorbia characias latex using ammonium sulfate precipitation and chromatography on DEAE-cellulose, hydroxylapatite and SP-Sephadex columns. The substrate specificity of the enzyme is typical of a plant peroxidase except that it shows no activity with indole-3-acetic acid. The pH optimum of the enzyme was 5.75 and the isoelectric point 7.4. The activation energy was 14 kcal/mol. The prosthetic group was shown to be ferriprotoporphyrin IX. Gel chromatography and PAGE indicate that the purified protein is composed of a single polypeptide chain having a MW of ca 48 000.     

The protein,lipid and carbohydrate composition of protein bodies from Lupinus angustifolius seeds

Protein bodies isolated from dehulled seeds of Lupinus angustifolius (cv New Zealand Bitter Blue) contained 73 % protein, of which 78 % were globul     

Isolation and properties of N-acetyllactosamine-specific lectins from nine erythrina species

Lectins from seeds of nine species of Erythrina have been purified by affinity chromatography on columns of lactose coupled to Sepharose and their properties compared with those of the lectin from Erythrina cristagalli. All lectins are glycoproteins of M, ca 60 000 composed of two identical or nearly identical subunits. They contain between 3–10% carbohydrates comprised of N-acetylglucosamine, mannose, fucose and xylose. The amino acid composition of all Erythrina lectins is very similar. The N-terminal amino acid is valine, with the exception of the lectin from E. flabelliformis in which it is alanine. To the extent tested, identities or near identities have been found in the N-terminal sequences (up to 15 residues in some cases) of the lectins. Hapten inhibition experiments of agglutination have shown that the lectins are specific for N-acetyllactosamine, this disaccharide being 10–30 times more inhibitory than D-galactose and 10–20 times more than N-acetyl-D-galactosamine. All lectins agglutinate human erythrocytes equally well, irrespective of blood type, at minimal concentrations of 5–20 μg/ml. Six of the lectins are also very effective in agglutinating rabbit erythrocytes and are mitogenic for human peripheral blood lymphocytes, whereas three of them are considerably weaker hemagglutinins for rabbit erythrocytes, and two of these are also very weak mitogens. Our results, while demonstrating striking similarities in the molecular properties and sugar specificity of all Erythrina lectins studied, suggest the existence of differences at or close to the carbohydrate-binding site.     

Purification and properties of asparagine synthetase from soybean root nodules

Asparagine synthetase was purified 240-fold from soybean (Glycine max (L.) Merr.) root nodules with a final recovery of 5% using Reactive Blue 2-crossed linked Agarose affinity gel chromatography. High levels of sulfhydryl protectants were required and the inclusion to glycerol and substrates in the extraction buffer helped to stabilize the enzyme. The final preparation had a specific activity of 3.77 mkat/kg protein when assayed at 30°C and was free of contaminating asparaginase activity. The enzyme had a broad pH maximum around pH 8.0 and apparent K_m values for the substrates aspartate, Mg · ATP, and glutamine were 1.24 mM, 0.076 mM and 0.16 mM, respectively. Ammonium ion could partially replace glutamine as the nitrogen donor. Initial velocity patterns yielded parallel inverse plots with all substrate pairs suggesting an overall ping-pong reaction mechanism. Product inhibition patterns provided evidence that glutamine was the first substrate to bind to the enzyme and asparagine was the last product released.     

The purification and properties of a thiaminase I enzyme from nardoo (Marsilea drummondii)

Fronds of the fern nardoo (Marsilea drummondii) contain a thiaminase I enzyme at very high levels of activity. Highest levels of enzyme activity were found in vigorously growing plant material. The thiaminase I has been purified to a final sp act value of 2.07 μkat/mg protein at 30° and pH 6.5. It was shown to have similar properties to thiaminase I enzymes purified from bracken fern, rock fern and freshwater mussels. These enzymes have MW values in the range 93 000–115 000, energies of activation of 14 000 cal mol, pH optima of 8–9 and are quite stable in the pH range 3 to 12 and to extended incubation at 55°. The temperature for 50 % denaturation is 60–65°. p-CMB, mersalyl acid and HgCl₂ (10^t-6 M) are potent inhibitors, but monoiodacetic acid (10^?4 M) has no effect. A wide range of heterocyclic bases, sulphydryl compounds, and amines, including the non-aromatic amines 6-aminohexanoic acid and ethanolamine, act as co-substrates in the thiaminase I reaction; however, their effectiveness is dependent on both their degrees of basicity and to some extent, their stereochemistry. When the co-substrate activity of a range of substituted anilines were compared, no correlation was found between the degree to which the base activates the reaction and the pK_b (or Hammett's sigma constant) of the base.     

Partial purification and properties of willardiine and isowillardiine synthase activity from Pisum sativum

The enzymic activity responsible for synthesis of willardiine and isowillardiine in pea seedlings has been extracted and partially purified. Fresh tissue, pulverized in liquid-N₂, was extracted in a phosphate buffer (pH 7) and subjected to fractional precipitation with ammonium sulphate. After desalting on Sephadex G-25 and concentration by ultrafiltration, the fraction containing the activity was chromatographed sequentially on DEAE-Sepharose CL-6B, DEAE-cellulose (DE 52) and Sephadex G-200. Electrophoretic separation in polyacrylamide gels was also used. A 120-fold purification was achieved but at no stage was there any indication of a separation of willardiine synthase activity from that of isowillardiine synthase. Both activities paralleled one another when the enzymic preparation was progressively denatured by subjecting it to gradually increasing temperatures. Similarly, ageing at 4° and at ?196° resulted in a parallel loss of activity. Both synthase activities were maximal at 7.8–7.9and the pH optimum curves were of closely similar shape. From the results described, it is concluded that a single enzyme of relatively low MW (ca 50 000) is responsible for the synthesis of both uracilylalanines. Studies of the alanylation of uracil using a pyridoxal-metal ion model-enzyme system are described.     

Purification and properties of lupin nodule glutamine synthetase

Glutamine synthetase (GS) from the cytoplasm of Lupinus luteus nodules was purified to apparent homogeneity using a final step of ADP-Sepharose affinity chromatography. Mercaptoethanol and divalent metals were essential to maintain the enzyme activity and keto compounds enhanced the stability during purification. From gel filtration a M, for the native enzyme of 347 000 was determined with subunits of 41 500 indicated by SDS-PAGE. The pH optima for the biosynthetic and transferase activities were 7.9 and 6.5 respectively. Mg²⁺-activated GS was strongly inhibited by Mn²⁺ and Ca²⁺; Co²⁺, while also inhibitory, allowed an alternate, more active form of GS after addition of glutamate. Activity was also inhibited by possible feedback inhibitors. The apparent K_m values for glutamate, NH₄⁺, ATP, glutamine, NH₂OH and ADP were 8.58 mM, 12.5 μM, 0.22 mM, 48.6 mM, 3.37 mM and 59.7 nM respectively.     

The isopentenyl isoflavone luteone as a pre-infectional antifungal agent in the genus Lupinus

The prenylated isoflavone luteone has been isolated from healthy leaves of Lupinus albus and 11 other lupin species. Evidence is presented that this compound occurs as a leaf surface constituent. In vitro tests indicate that luteone and a second unidentified isoflavone frorn L. albus possess antifungal activity sufficient to support their proposed role as pre-infectional resistance factors. No evidence was obtained to suggest that phytoalexins were produced by the fungus-infected leaves of L. albus.     

The purification of methionine sulfoxide reductase from Escherichia coli

A sensitive assay, based on the acylation of tRNA^Met, has been developed to measure the enzymatic reduction of methionine sulfoxide to methionine. Using this assay, methionine sulfoxide reductase has been purified to near homogeneity from extracts of Escherichia coli.     

Diamine oxidase from Lens esculenta seedlings: Purification and properties

A diamine oxidase (DAO) (EC 1.4.3.6) has been purified to homogeneity from lentil seedlings. The purified protein has a MW of 154 000 and is composed of two apparently identical subunits. It contains two CU²⁺ atoms and one carbonyl-like group per mol. The purified enzyme is pink-red in concentrated solution and shows a broad, well-defined, absorption band in the visible region centered at 498 nm. The ESR spectrum is typical of Cu²⁺ in a tetragonal symmetry. The enzyme oxidizes only aliphatic diamines and spermidine with formation of the corresponding aldehydes, hydrogen peroxide and ammonia. Putrescine and cadaverine are oxidized most rapidly and the oxidation rate decreases when longer diamines are tested.     

Comparative properties of ferredoxins from a marine and freshwater species of Porphyridium

Ferredoxins were isolated from the freshwater red alga Porphyridium aerugineut, and from Porphyridium cruentum, a related marine species. A sin     

The purification and properties of a fibrinolytic neutral metalloendopeptidase from Streptococcus faecalis

A protease has been isolated by affinity chromatography from culture filtrates of a strain of Streptococcus faecalis previously shown to produce a flbrinolytic enzyme. The pH optimum, molecular weight, metal ion chelator sensitivity, and peptidase specificity place this enzyme in the class of bacterial neutral metalloendopeptidase typified by thermolysin and the Bacillus subtilis neutral proteases. Differences with respect to chemical modification and thermal stability exist between the S. faecalis enzyme and the latter proteases. The S. faecalis enzyme (designated EM 19000) renders fibrinogen incoagulable by degradation of the B (β) chains.