Nitrate Reduction in Different Grass Species

Optimal extraction conditions, assay conditions, and levels of nit rate reductase activity (NRA) were determined for eight forage grass species adaptable to growing conditions in western North Dakota. Optimal pH for extraction of the enzyme nitrate reductase (NADH: nitrate oxidoreductase) for these species ranged from 7.0 to 9.5, whereas assay pH was 7.6 in all eight species. Substrate concentrations ranged from 1.0 to 10.0 mM for maximum NRA with higher concentrations (100 mM) significantly inhibiting NRA. The enzyme was NADH₂ (0.1 to 0.2 mM for maximum activity) specific. Enhancement of maximum activity with the addition of cysteine during extraction was species dependent; six species required high cysteine concentrations between 5 mM and 10 mM and one species required only a 2.5 mM concentration. The degree of sulfhydryl protection offered by cysteine also varied. Comparisons were made between in vivo and in vitro assay methods. Ratios of in vitro to in vivo NRA ranged from 2.2. to 10.8. Use of bovine serum albumin as a protein stabilizer during extraction increased the measurable NRA in some species. Applications of nitrate reductase assay techniques to field work will be discussed.     

Microphotometric determination of enzymes in brain sections

Summary A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM d-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN₃, 10 mM MgCl₂, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 m. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1, 2-2)     

Induction of Nitrate Reductase and Peroxidase Activity in the Seedlings of Normal and High Lysine Maize

Steady state levels of in vivo nitrate reductase activity in the endosperm, scutella, roots and shoots of maize seedlings were higher in normal maize than those in high lysine maize. Activity of peroxidase in the roots, however, was higher in the high lysine cultivar. The nitrate reductase activity increased with the supply of nitrate in all parts of the seedlings of both cultivars, although the rates of increment in the endosperm were lower than those in scutella, roots and shoots. In relation to substrate concentration, a saturation was achieved at 5 to 10 mM of nitrate except in the endosperm, where activity increased slowly up to 100 mM at least. Final levels of enzyme activity were significantly higher in the scutella of normal than in that of high lysine seedlings. In vitro enzyme activity in the roots also increased with the supply of nitrate in both cultivars, reaching maximum at 5 to 10 mM nitrate.     

Microphotometric determination of enzymes in brain sections. II. GABA transaminase

Summary The tetrazolium salt procedure of van Gelder (1965) for the demonstration of GABA transaminase (GABAT; the most important GABA degrading enzyme) was adapted for microphotometric measurements of GABAT activities in brain sections using the hippocampus of rats as selected brain region. The final incubation medium consisted of 50 mM GABA, 5 mM α-ketoglutarate, 7 mM NAD, 10 mM sodium azide, 6 mM nitroblue tetrazolium chloride, 20 mM malonate and 15% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 8.0. There was a linear relationship between GABAT activity and section thickness up to 14 μm and between GABAT activity and reaction time at least up to 20 min (kinetic and end-point measurements). Phenazine methosulfate as an exogenous electron carrier and pyridoxal-5-phosphate as coenzyme of GABAT did not enhance the demonstrable GABAT activities, whereas sodium azide as a blocker of the respiratory chain resulted in an increase of demonstrable enzyme activities. A coreaction of succinate dehydrogenase was excluded by the use of malonate (competitive inhibitor). Using the incubation medium described GABAT activities were demonstrated via the endogenous enzymes succinic semialdehyde dehydrogenase and NADH tetrazolium reductase which were shown to be not rate limiting and seems to be similarly localized as GABAT. Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2)     

High polyphenol oxidase activity and low titratable acidity in browning bamboo tissue culture

Summary Tissue browning that frequently results in the early death of bamboo shoots in vitro correlated directly with polyphenol oxidase (PPO, EC 1.10.3.1) activity and inversely with titratable acidity. It was unrelated to the level of endogenous phenols. During the course of culture, timing of PPO activity paralleled that of explant browning. Browning was highest among shoots cultured in a medium of pH 8, which was consistent with the pH optinum of the bamboo enzyme. The pH optimum was first determined with the crude enzyme, then verified with two purified isozymes. Stability of the bamboo PPO was also highest at pH 10. PPO activities of the severely browning Dendrocalamus latiflorus, the moderately browning Phyllostachys nigra, and the relatively non-browning Bambusa oldhamii were inhibited strongly by ascorbic acid, cysteine, sodium diethyldithiocarbamate, and sodium sulfite. But characterization of bamboo PPO according to enzyme inhibitors was not possible because enzyme extracts of the three species gave varied responses to the traditional substances. Nutrient medium addenda of some PPO inhibitors, namely ascorbic acid, cysteine, kojic acid, and thiourea, mainly enhanced browning. However, ferulic acid at 3 mM and lower concentrations reduced the number of brown shoots per culture, although not the percentage of cultures that browned. Polyvinylpyrrolidone failed completely to suppress browning. The two purified isozymes showed different temperature optima for PPO activity: 60°C and 65°C. The purified isozymes displayed a substrate preference for dopamine, or a cathecol oxidase characteristics.     

1,3-β-d-Glucan synthase ofNeurospora crassa: Partial purification and characterization of solubilized enzyme activity

1,3-β-d-Glucan synthase activity ofNeurospora crassa was rendered soluble by treatment of crude protoplast lysates with 0.1% 3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 1 mM dithiothreitol, 200 mM inorganic phosphate, 10 μM GTP, and 600 mM glycerol. Approximately 50% of enzyme activity was solubilized; soluble enzyme activity was purified 5.6-fold with a net 38% recovery by sucrose gradient density sedimentation. Partially purified enzyme activity had a half-life of 60 ± 10 h at 4°C, aK_m,app of 0.75 ± 0.05 mM, and a V_max,app of 35 ± 1 enzyme units/mg protein.     

Purification and Some Properties of Acid Invertase of Persimmon Fruits

Single cells were prepared from mesocarp tissue of ripe persimmon (Diospyros kaki cv. Fuyu) fruits, and inter- or intracellular localization of acid invertase (AI, EC 3.2.1.26) was studied. AI was localized in the intercellular fraction (cell wall fraction). AI was isolated and purified from the cell wall fraction of ripe persimmon fruits by column chromatography on SE-53 cellulose and Toyopearl HW 55F. The specific activity of purified AI was 570 units per mg protein at 30°C. The molecular mass of AI was estimated to be 44 kDa by gel filtration over Sephacryl S-200 and 70 kDa by SDS–PAGE. The optimum pH of the activity for sucrose was 4.25. The purified enzyme hydrolyzed sucrose and raffinose but not melibiose. The enzyme had a K_m of 3.2 mM for sucrose and a K_m of 2.6 mM for raffinose. Silver nitrate (5 μM), HgCI₂ (2 μM), p-chloromercuribenzoate (100mM), pyridoxamine (10mM), and pyridoxine (2.5mM) inhibited AI activity by 95, 85, 100, 41, and 300%, respectively.     

Philibertain g I, the Most Basic Cysteine Endopeptidase Purified from the Latex of Philibertia gilliesii Hook. et Arn. (Apocynaceae)

A new papain-like cysteine peptidase isolated from latex of Philibertia gilliesii Hook. et Arn., Apocynaceae (formerly Asclepiadaceae) has been purified and characterized. The enzyme, named philibertain g I, is the most basic component present in latex extracts and was purified by acetone fractionation followed by cation exchange chromatography (SP-Sepharose HR) using FPLC system. Homogeneity was confirmed by SDS-PAGE and mass spectroscopy (MS). Molecular mass of the enzyme was 23,530 Da (MALDI-TOF MS), its isoelectric point was >10.25, and maximum proteolytic activity (casein) was achieved at pH 7–8. The new protease was inhibited by E-64 a cysteine peptidases inhibitor. K_m was 0.15 mM, using PFLNA as substrate. The N-terminal sequence of philibertain g I (LPASVDWRKEGAVLPIRHQGQCG) was compared with those of twenty plant proteases. Philibertain g I showed the higher degree of identity (73%) with caricain, one of the Carica papaya endopepetidases.     

Further characterization of the two catalases inEscherichia coli

Crude extracts of wild-typeEscherichia coli contain two catalase species that separate on native polyacrylamide gels. The slow-migrating enzyme (HPII) has two pH optima of activity (at pH 6.8 and 10.5), is activated at 70°C, is sensitive to inhibitory by 3-amino-1,2,4-triazole and has Km values of 18.2 mM at pH 6.8 and of 10 mM at pH 10.5. The fast-migrating enzyme has a single pH optimum of 6.8 and is composed of two isozymes (HPI-A and HPI-B). Its activity is labile at 70°C, it is relatively resistant to inhibition by 3-amino-1,2,4-triazole and has a Km value of 3.7 mM.     

Expression Optimization and Biochemical Characterization of a Recombinant γ-Glutamyltranspeptidase from Escherichia coli Novablue

A truncated Escherichia coli Novablue γ-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His₆-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 °C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 °C and 9, respectively. The apparent K _m and V _max values for γ-glutamyl-p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μM and 53.7 × 10⁻³ mM min⁻¹, respectively. The synthesis of L-theanine was performed in a reaction mixture containing 10 mM L-Gln, 40 mM ethylamine, and 1.04 U His₆-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained.     

Light and ethylenediamine tetraacetic acid mediated differential effects of ammonium on nitrate reductase activity in maize seedlings

Abstract Effect of ammonium on in vivo activity of nitrate reductase in roots, shoots and leaves of maize (Zea mays L.) seedlings was studied in relation to light/dark conditions and EDTA supply. Supply of 5 mM (NH₄)₂SO₄ increased the steady state level of enzyme only in leaves and in light, while it had no effect in roots and shoots and in the dark. The substrate induction of enzyme was also little affected by 1 to 10 mM (NH₄)₂SO₄ in roots and shoots. In the leaves the activity in the dark was either inhibited (minus EDTA) or stimulated (plus EDTA) by 5 to 10 mM (NH₄)₂SO₄. The activity was stimulated in the light also in the presence of EDTA at higher concentrations of ammonium. When different concentrations of ammonium were supplied without any exogenous nitrate in the light, the enzyme activity increased at low concentration and was either inhibited or unaffected at higher concentrations depending upon the tissue used. Supply of EDTA with ammonium modified its effect to some extent. It is suggested that the effect of ammonium on nitrate reductase activity depends upon the tissue used and the effective concentration of the ammonium.     

Purification and Characterization of a Novel (R)-Imine Reductase from Streptomyces sp. GF3587

The (R)-imine reductase (RIR) of Streptomyces sp. GF3587 was purified and characterized. It was found to be a NADPH-dependent enzyme, and was found to be a homodimer consisting of 32 kDa subunits. Enzymatic reduction of 10 mM 2-methyl-1-pyrroline (2-MPN) resulted in the formation of 9.8 mM (R)-2-methylpyrrolidine ((R)-2-MP) with 99% e.e. The enzyme showed not only reduction activity for 2-MPN at neutral pH (6.5–8.0), but also oxidation activity for (R)-2-MP under alkaline pH (10–11.5) conditions. It appeared to be a sulfhydryl enzyme based on the sensitivity to sulfhydryl specific inhibitors. It was very specific to 2-MPN as substrate.     

Proline metabolism in response to nitrogen deficiency in French Bean plants (Phaseolus vulgaris L. cv Strike)

The objective of the present work was to determine the impact ofnitrogen deficiency on proline metabolism in French Bean plants(Phaseolus vulgaris L. cv. Strike). The nitrogen wasapplied to the nutrient solution in the form of NH₄NO₃ at1.45 mM (N1), 2.90mM (N2) and 5.80mM (N3, optimal level). Our results indicateNdeficiency is characterised by a decline in proline accumulation both in theroot and leaves, fundamentally because proline degradation is encouraged by thestimulation of the enzyme proline dehydrogenase. By contrast, under conditionsof adequate N (N3), proline levels rise due to the action of ornithine,suggesting predominance of the ornithine pathway over the glutamine pathway, inaddition to the inhibition of proline dehydrogenase activity.     

Preparation of immobilized soybean β-amylase on porous cellulose beads and continuous maltose production

Immobilized soybean β-amylase was prepared by using porous cellulose beads. The expressed activity of the β-amylase–cellulose beads conjugated below 35 mesh was 59–69% of the initial activity and the protein content was 10–13%. General properties of the conjugate were almost identical with those of the native enzyme except for the K_m value. The K_m value of the conjugate was 40mM and the K_m value of the native enzyme was 0.6mM. This large difference was probably caused by pore structure, i.e., a pore diffusion problem. The film diffusion problem occurred at the flow rate below a linear velocity of 3 cm/min. Maximum maltose contents of the hydrolyzates prepared by the conjugate and the native enzyme were 69 and 71%, respectively. After a continuous column operation at 50°C for 17 days, the activity of the column was 60% of the activity. The half-life of the column at 40°C was 40 days.     

Characterization of glutamine synthetase in the apple

Glutamine synthetase (l -glutamate: ammonia ligase, ADP-forming, EC 6.3.1.2) in bark tissue of the apple (Malus domestica Borkh. cv. Golden Delicious) was partially purified and characterized. The Mn²⁺- and Mg²⁺-dependent activities were maximal at pH 7.2 and 7.5, respectively. The enzyme was almost completely inactivated within two weeks at 0°C. Both Mg²⁺ and β-mercaptoethanol were effective in stabilizing the enzyme during storage. The enzyme was protected from thermal inactivation at 60°C by the addition of Mg²⁺ and ATP. One-tenth mM phenylmercuric acetate inhibited the Mg²⁺-dependent activity by 50%. Equimolar dithiothreitol protected the enzyme from this inactivation. The K_m values of the enzyme were 0.27, 7.35, and 0.69 mM for ATP, glutamate, and NH₂OH, respectively. The constant for NH⁺₄ was an order of magnitude higher in the presence of Mn²⁺ than Mg²⁺. When the amino acids were externally added to the reaction mixtures, the measurement of P_i exhibited a higher degree of enzyme inhibition than the measurement of γ-glutamyl monohydroxamate (GHA). Ten mM histidine inhibited the Mg²⁺- and Mn²⁺-dependent activities by 26 and 45% respectively. Twenty mM aspartate (d,l -form) inhibited the enzyme 30% in the presence of either Mg²⁺ or Mn²⁺. Aspartate (Mg²⁺-dependent) and histidine (Mn²⁺-dependent) inhibited the enzyme competitively with respect to glutamate, the estimated inhibition constants being 17.6 and 1.6 mM, respectively. At 10 mM, amino acids such as tryptophan, arginine, alanine and citrulline inhibited enzyme activity from 1 to 18%. Glutamine stimulated the Mg²⁺-dependent activity 25% at 25 mM when GHA was measured. Glutamine above 32 mM inhibited the enzyme.     

Proline-Dehydrogenase from Pumpkin (Cucurbita moschata) Cotyledons

A NAD specific proline-dehydrogenase was found in pumpkin (Cucurbita moschata Poir. cv. Dickinson Field) which oxidized proline to Δ¹-pyrroline-5-carboxylate. NADP did not substitute for NAD and L-proline-methyl-ester and thiazolidine-4-carboxylate were substrates in the reaction, at a rate of 107% and 33% respectively, of the rate with L-proline. Pumpkin cotyledons contained the bulk of the enzyme activity with 90% of the activity being in the soluble fraction. Proline-dehydrogenase, which was not treated at high temperature, was stable at –10°C for 4 months in the presence of high ammonium sulfate concentration. The Michaelis constant for NAD was 2.2 mM and for L-proline was 2.5 mM. At 5 mM NADP, a 40% non-competitive inhibition of proline-dehydrogenase was obtained, while 50 μM NADP was sufficient to induce 20% inhibition.     

Evidence of morphology-specific isozymes inCandida albicans

S-Adenosylmethionine (SAM) synthetase of yeast and hyphal-phase cells of the dimorphic fungusCandida albicans was characterized by kinetic analysis and response to inhibitors. The enzyme from yeast-phase cells has a Km of 0.17 mM for methionine, 0.14 mM for ATP, and is inhibited (in vitro) by dimethyl-sulfoxide, methionine sulfone, and methionine sulfoxide. The hyphal-phase SAM synthetase has a Km of 0.06 mM for methionine, 0.02 mM for ATP, and its activity (in vitro) is enhanced by the substances that inhibit the yeast-phase enzyme. These data strongly suggest that isozymes of SAM synthetase are present inC. albicans and that they are possibly morphology specific. In vivo studies revealed that synthesis of the enzyme is repressed by the addition of methionine to the growth medium and that specific activity of the enzyme increases when intracellular SAM levels are lowered. In addition, it was shown that the increase in specific activity seen during yeast hypha morphogenesis and in yeast cells grown in a methionine-free medium involves de novo protein synthesis.     

A Highly N-Glycosylated Chitin Deacetylase Derived from a Novel Strain of Mortierella sp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2–4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1–10 mM Co²⁺ and strongly inhibited by 10 mM Cu²⁺. It required at least two GlcNAc residues for catalysis. When (GlcNAc)₆ was used as substrate, K _m and V _max were determined to be 1.1 mM and 54.6 μmol min^?1 respectively.     

Extracellular Laccase Produced by an Edible Basidiomycetous Mushroom,Grifola frondosa: Purification and Characterization

A major laccase isozyme (Lac 1) was isolated from the culture fluid of an edible basidiomycetous mushroom, Grifola frondosa. Lac 1 was revealed to be a monomeric protein with a molecular mass of 71 kDa. The N-terminal amino acid sequence of Lac 1 was highly similar to those of laccases of some other white-rot basidiomycetes. Lac 1 showed the typical absorption spectrum of a copper-containing enzyme. The enzyme was stable in a wide pH range (4.0 to 10.0), and lost no activity up to 60 °C for 60 min. The optimal pH of the enzyme activity varied among substrates. The K _m values of Lac 1 toward 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol, catechol, and 3,4-dihydroxy-L-phenylalanine were 0.0137 mM, 0.608 mM, 0.531 mM, 2.51 mM, and 0.149 mM respectively. Lac 1 activity was remarkably inhibited by the chloride ion, in a reversible manner. Lac 1 activity was also inhibited by thiol compounds.     

Thermal Inactivation and Product Inhibition of Aspergillus terreus CECT 2663 α-L-Rhamnosidase and Their Role on Hydrolysis of Naringin Solutions

The kinetics of thermal inactivation of A. terreus α-rhamnosidase was studied using the substrate p-nitrophenyl α-L-rhamnoside between 50°C and 70°C. Up to 60°C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60°C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60°C. Hydrolysis of naringin reached 99% with 1% naringin solution, although the hydrolysis degree of naringin was only 40% due to products inhibition when the initial concentration of flavonoid was 10%. The experimental results fitted an equation based on the integrated Michaelis-Menten's, including competitive inhibition by products satisfactorily.