Nitrogenase activity in Rhodopseudomonas sulfidophila

The marine purple nonsulfur bacterium, Rhodopseudomonas sulfidophila, strain W4, was capable of photosynthetic growth on dinitrogen and malate. Higher growth rates were observed when either glutamate or ammonia replaced dinitrogen as nitrogen source and when bicarbonate was omitted from the culture medium. Although ammonia was released from cells growing on malate and N₂, no nitrogenase activity could be detected unless -ketoglutarate was added to the culture medium. No nitrogenase activity was found in cultures grown in the presence of NH ₄ ⁺ . In cultures grown on glutamate as nitrogen source, nitrogenase and hydrogenase activities were found to be 5.4 nmol C₂H₂ reduced · min^-1 · mg^-1 dry weight and 50 nmol methylene blue reduced · min^-1 · mg^-1 dry weight respectively. Such activities are significantly lower than those observed for other members of the Rhodospirillaceae e.g. Rhodopseudomonas capsulata. However, the hydrogenase activity would be sufficient to recycle all H₂ produced by nitrogenase. It was indeed observed that growing cells did not evolve molecular hydrogen during photoheterotrophic growth and that H₂ stimulated nitrogenase activity in resting cells of R. sulfidophila. The nitrogenase from this bacterium proved to be extremely sensitive to low concentrations of oxygen, half-inhibition occurring at between 1–1.5% O₂ in the gas phase, depending on the bacterial concentration. Light was essential for nitrogenase activity. No activity was found during growth in the dark under extremely low oxygen concentrations (1–2% O₂), which are still sufficient to support good growth. Resting cell suspensions prepared from such cultures were unable to reduce acetylene upon illumination. Optimum nitrogenase activities were broadly defined over the temperature range, 30–38°C, and between pH 6.9 and 8.0. The results are discussed in comparison with the non-marine purple nonsulfur bacterium, R. capsulata, which somewhat resembles R. sulfidophila.     

The induction of nitrogenase activity in Rhizobium by non-legume plant cells

Summary Nitrogen fixation was induced in a strain of cowpea rhizobia, 32Hl, when it was grown in association with cell cultures of the non-legume, tobacco (Nicotiana tabacum). Rhizobia grown alone on the various media examined did not show nitrogenase activity, indicating the involvement of particular plant metabolites in nitrogenase induction. Nitrogenase activity, as measured by C₂H₂ reduction, was maximized at an O₂ concentration of 20% and at an assay temperature of 30°C, the conditions under which the plant cell-rhizobia associations developed. Glutamine, as a nitrogen source, could be replaced by other organic nitrogen sources, but NH₄ ⁺ and NO₃ ^- repressed nitrogenase activity. Nitrogenase activity induced in rhizobia when cultured adjacent to, but not in contact with, the plant cells could be stimulated by providing succinate in the medium. At least 12 other strains of rhizobia also reduced C₂H₂ in association with tobacco cells; the highest levels of activity were found among cowpea strains.     

Enhancement of specific nitrogenase activity inAzospirillum brasilense andKlebsiella pneumoniae,inhibition inRhizobium japonicum under air by phenol

Specific nitrogenase activity inAzospirillum brasilense ATCC 29145 in surface cultures under air is enhanced from about 50 nmol C₂H₄·mg protein^-1·h^-1 to 400 nmol C₂H₄ by the addition of 1 mM phenol. 0.5 and 2 mM phenol added increase the rate 5-fold and 4-fold. This enhancement effect is observed only between 2 and 3 days after inoculation, with only a small reduction of the growth of the cells by the phenol added. In surface cultures under 1% O₂, nitrogenase activity is slightly reduced by the addition of 1–0.01 mM phenol. Utilization of succinate is enhanced during the period of maximum enhancement of nitrogenase activity by 60% by addition of 1 mM phenol. The cells did not produce¹⁴CO₂ from [U-¹⁴C] phenol, neither in surface cultures nor in liquid cultures and less than 0.1% of the phenol was incorporated into the cells. A smaller but significant enhancement of nitrogenase activity by about 100% in surface cultures under air was found withKlebsiella pneumoniae K 11 after addition of 1 mM phenol. However, inRhizobium japonicum 61-A-101 all phenol concentrations above 0.01 mM reduced nitrogenase activity. With 1 mM phenol added activity was reduced to less than 10% with no effect on the growth in the same cultivation system. With thisRhizobium japonicum strain significant quantities of phenol (25 mol in 24 h by 2·10¹² cells) were metabolized to¹⁴CO₂, with phenol as sole carbon source. WithAzospirillum brasilense in liquid culture under 1% and 2% O₂ in the gas phase, no enhancement of nitrogenase activity by phenol was noticed.     

Upper temperature limits for growth and diazotrophy in the thermophilic cyanobacterium HTF Chlorogloeopsis

The effect of temperature and oxygen on diazotrophic growth of the thermophilic cyanobacterium HTF (High Temperature Form) Chlorogloeopsis was investigated using cells grown in light-limited continuous culture at a dilution rate of 0.02 h^-1. Diazotrophy was more sensitive to elevated temperatures than growth with combined nitrogen. The maximum temperature for growth of cultures gassed with CO₂-enriched air was more than 55 °C but less than 60 °C with N₂ as the sole nitrogen source, but between 60°C and 65°C when nitrate was present in the medium. The effect of temperature on nitrogenase activity, photosynthesis and respiration in the dark was determined using cells grown at 55°C. Maximal rates of all three processes were observed at 55°C and rates at 60°C during shortterm incubations were not less than 75% of the maximum. However, nitrogenase activity at 60°C was unstable and decayed at a rate of 2.2 h^-1 under air and at 0.3 h^-1 under argon. Photosynthesis and respiration were more stable at 60°C than anoxic nitrogen fixation. The upper temperature limits for diazotrophic growth thus seem to be set by the stability of nitrogenase.Abbreviations chl chlorophyll a - DCMU N-(3,4-dichlorophenyl) N,N-dimethylurea - Taps N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid     

Purification and characterization of a thermostable and acid-stable phytase from Pichia anomala

Phytase of Pichia anomala was purified to near homogeneity by a two-step process of acetone precipitation followed by anion exchange chromatography using DEAE-Sephadex. The enzyme had a molecular weight of 64 kDa. It was optimally active at 60 °C and pH 4.0. This enzyme was found to be highly thermostable and acid-stable, with a half life of 7 and 8 days at 60 °C and pH 4.0 respectively. At 80 °C, the half life of phytase could be increased from 5 to 30 min by the addition of materials such as sucrose, lactose and arabinose (10% w/v). The enzyme exhibited a broad substrate specificity, since it acted on p-nitrophenyl phosphate, ATP, ADP, glucose-6-phosphate besides phytic acid. The K _m value for phytic acid was 0.20 mM and V _max was 6.34 mol/mg protein/min. There was no requirement of metal ions for activity. SDS was observed to be highly inhibitory to phytase activity. Sodium azide, DTT, -mercaptoethanol, EDTA, toluene, glycerol, PMSF, iodo-acetate and N-bromosuccinimide did not show inhibitory activity. The enzyme was inhibited by 2,3-butanedione, indicating the involvement of arginine residues in catalysis. Phytase activity was not inhibited in the presence of inorganic phosphate upto 10 mM. The shelf life of the enzyme was 6 months at 4 °C and there was no loss in the activity on lyophilization. Very few studies have been done on purification of yeast phytases. This is the first report on purification and characterization of phytase from P. anomala. The enzyme is unique in being thermostable, acid-stable, exhibiting broad substrate specificity and in not requiring metal ions for its activity. The yeast biomass containing phytase appears to be suitable for supplementing animal feeds to improve the availability of phosphorus from phytates.     

Mutants of Rhizobium capable of fixing N2 in the free-living condition

Six mutant strains of Rhizobium were isolated after UV treatment which could exhibit nitrogenase activity in Burk's N-free medium without any supplement. The activity ranged between 99.5 and 113 nmol/mg cell dry weight and hour. Two of the parent strains belonged to soybean, and one each to mungbean and Sesbania sp. Both the parent and mutant strains exhibited nitrogenase activity in CS 7 medium. One of the mutants retained its capacity to produce nodules on soybean roots.List of Abbreviations C.D. Critical difference - EMS ethylmethane sulphonate - NTG N-methyl-N-nitro, N-nitrosoguanidine     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Nitrogenase — hydrogenase relationships in Rhizobium japonicum

The conditions necessary for coordinate derepression of nitrogenase and O₂-dependent hydrogenase activities in free-living cultures of Rhizobium japonicum were studied. Carbon sources were screened for their ability to support nitrogenase, and then hydrogenase activities. There was a positive correlation between the level of nitrogenase and corresponding hydrogenase activities among the various carbon substrates. The carbon substrate -ketoglutarate was able to support the highest levels of both nitrogenase and hydrogenase activities. When cells were incubated in -ketoglutarate-containing medium, without added H₂ but in the presence of acetylene (to block H₂ evolution from nitrogenase) significant hydrogenase activity was still observed. Complete inhibition of nitrogenase-dependent H₂ evolution by acetylene was verified by the use of a Hup^- mutant. Hydrogen is therefore not required to induce hydrogenase. The presence of 10% acetylene inhibited derepression of hydrogenase. Constitutive (Hup^c) mutants were isolated which contained up to 9 times the level of hydrogenase acitivity than the wild type in nitrogenase induction medium. These mutants did not have greater nitrogenase activities than the wild type.This is contribution number 1254 from the Department of Biology and the McCollum-Pratt Institute Abbreviations: -Ketoglutarate-containing medium (LOKG) and pre-adaptation medium (SRM) as described in Materials and methods     

N2-fixation in Erwinia herbicola

Four from 18 strains of Erwinia herbicola tested had nitrogenase activity and grew with N₂ as sole source of nitrogen under strict anaerobic conditions with a doubling time of 20–24 h. Nitrogenase activity started only 96–120 h after transfer to a special medium maintained under anaerobic conditions. A ten fold increase in protein per culture found after the maximum nitrogenase activity of 80–130 nmol C₂H₄. mg protein^-1·min^-1 was accompanied by a fall in pH of the medium (20 mM phosphate buffer and in 125 mM Tris-buffer) from pH 7.2 to 5.4 or less, but only to 6.8 in 100 mM phosphate buffer. In all cases we found a sharp curtailing of nitrogenase activity 48 h after the maximum. The bacteria utilized only 35–50% of the nitrogen fixed for growth. Erwinia herbicola strains differed from two strains of Enterobacter agglomerans in being unable to fix nitrogen on agar surfaces exposed to air. Specific nitrogenase activity in Erwinia herbicola is compared with data reported for other Enterobacteriaceae and is found to be higher than that reported for Klebsiella pneumoniae, Enterobacter cloacae or Citrobacter freundii.     

Calcium requirement in nitrogen fixation in the cyanobacterium Synechococcus RF-1

Under diurnal 16/8-h light-dark cycles, ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) at 1 mM completely blocked the appearance of rhythmic N₂-fixing activity in Synechococcus RF-1. Ca²⁺ at 2 mM, when supplied either together with or several hours after the EGTA application, restored the nitrogenase activity, whereas, when Ca²⁺ was supplied several hours later, the peak of nitrogenase activity was shifted from the dark to the light period in which the activity is normally suppressed. Sr²⁺ also reversed the inhibition by EGTA, but only partially. When O₂ in the gas phase above the culture was below 1%, the inhibition of nitrogenase activity by EGTA was reduced to less than 20% of the control value without EGTA. Thus Ca²⁺ appears to be required by the cell to protect its nitrogenase from inactivation by O₂. In media without EGTA, a close correlation between nitrogenase activity and concentrations of Ca²⁺ was also observed.Abbreviation EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings

During early development (up to 18 d after sowing) of nodules of an effective cowpea symbiosis (Vigna unguiculata (L.) Walp cv. Vita 3: Rhizobium strain CB756), rapidly increasing nitrogenase (EC 1.7.99.2) activity and leghaemoglobin content were accompanied by rapid increases in activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), enzymes of denovo purine synthesis (forming inosine monophosphate) xanthine oxidoreductase (EC 1.2.3.2), urate oxidase (EC 1.7.3.3), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and led to increased export of ureides (allantoin and allantoic acid) to the shoot of the host plant in the xylem. Culturing plants with the nodulated root systems maintained in the absence of N₂ (in 80 Ar: 20 O₂, v/v) had little effect on the rates of induction and increase in nitrogenase activity and leghaemoglobin content but, in the absence of N₂ fixation and consequent ammonia production by bacteroids, there was no stimulation of activity of enzymes of ammonia assimilation or of the synthesis of purines or ureides. Addition of NO ₃ ^- (0.1–0.2 mM) relieved host-plant nitrogen deficiency caused by the Ar: O₂ treatment but failed to increase levels of enzymes of N metabolism in either the bacteroid or the plant-cell fractions of the nodule. Premature senescence in Ar: O₂-grown nodules occurred at 18–20 d after sowing, and resulted in reduced levels of nitrogenase activity and leghaemoglobin but increased the activity of hydroxybutyrate oxidoreductase (EC 1.1.1.30).     

Two novel extracellular cholesterol oxidases of Bacillus sp. isolated from fermented flatfish

Two novel extracellular cholesterol oxidases designated CO1 and CO2, from Bacillus sp. SFF34, were purified 5.6 and 5.9-fold giving Mr values of 36 and 37 kDa. The optimum temperature for the activity was 60 °C (CO1) and 40 °C (CO2), and the optimum pH was 6.25 (CO1) and 6 (CO2) over 30 min reaction time. The apparent K _m values for cholesterol were 6.76 mM (CO1) and 4.50 mM (CO2). Both the enzymes could oxidize 5-cholestane, 5-cholestane-3-ol-7-one, coprostane, dihydrocholesterol, hecogenin, -sitosterol and stigmasterol.     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Reactivation by chloride of hill activity in heat- and tris-treated thylakoid membranes from Beta vulgaris

Hill activity (photoreduction of 2,6,dichlorophenol indophenol) of heat inactivated (40°C, 3 min) and Tris-washed (0.8M, pH 8.3) thylakoids of Beta vulgaris (beet-spinach) was partially restored if they were incubated with 150 mM MgCl₂ prior to the assay. Mg(NO₃)₂ or MgSO₄ were unable to restore this activity. The extent of this reactivation was dependent upon the degree of inactivation by heating and upon the composition of the isolation and the resuspension buffer used during the heat treatment. Washing of heat-treated thylakoids with phosphate-EDTA buffer prior to incubation with MgCl₂ did not affect the extent of this reactivation. Chloride ions seem to be required for the reactivation of Hill activity damaged either by heat or by Tris.Most commonly used chloroplast isolation and resuspension media, except for Tris-HCl as resuspension medium, were suitable for restoration of Hill activity in heat-damaged thylakoids by preincubation with 150 mM MgCl₂ prior to the assay. Pretreatment with MgCl₂ stimulated Hill activity in Tris-treated and heat-damage thylakoids if phosphate buffer was used for their resuspension. However, the same pretreatment inhibited Hill activity in unheated thylakoids isolated in Tris medium and resuspended in the same medium. On the other hand, MgCl₂ pretreatment induced restoration of the Hill activity of the heated thylakoids when Tricine or Hepes was used as the resuspension medium. It appears that the presence of Tris somehow hampers the Cl^– induced reactivation. The stimulation of Hill activity by MgCl₂ treatment in unheated (control) thylakoids is possibly induced by Mg²⁺ ions and not by Cl^– ions.Abbreviations Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1. 1-dimethyl-urea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2 hydroxyethyl piperazine-N, 2 ethano-sulfonic acid - HT heat-treated - PS II photosystem II - Tricine N-tri (hydroxymethyl) methyl glycine - Tris Tris-(hydroxymethyl) amino-methane     

The influence of metal cations and pH on the heat sensitivity of photosynthetic oxygen evolution and chlorophyll fluorescence in spinach chloroplasts

The heat-sensitivity of photosynthetic oxygen evolution of thylakoids isolated from spinach increases by increasing the pH above neutral value. The temperature for inactivation (transition temperature) is lowered from about 45° C (pH 6.0–7.4) to 33°C (pH 8.5). Similar results are obtained with intact chloroplasts. At pH 7.0 the transition temperature of washed thylakoids decreases by lowering the salt concentration below 20 mM with monovalent cations (Li⁺, Na⁺, K⁺) and below 3–4 mM with divalent cations (Mg²⁺, Ca²⁺, Sr²⁺). Illumination decreases the heat-sensitivity of oxygen evolution in intact chloroplasts, but even increases the heat-sensitivity in uncoupled chloroplasts. In intact chloroplasts the transition temperature of the heat-induced rise in chlorophyll fluorescence yield (F_o; see Schreiber and Armond 1978) decreases from 44° C to 38° C when the pH of the suspending medium is increased from 6.5 to 8.5. At 20° C, F_o is almost insensitive to pH (6.0–8.5). At 40° C, however, F_o is constant between 6.0 and 7.0, but strongly increases by increasing the pH above neutral value. The results are discussed in terms of a close relation between electrostatic forces at the thylakoid membrane and thermal sensitivity of photosynthetic apparatus. It is suggested that the heat-sensitivity of the photosystem II complex partially depends on the ionization state of fixed groups having alkaline pK. The packed volume of thylakoids suspended in a low salt medium increases when the temperature is increased above 30° C (pH 7.0) and above 20° C (pH 8.0), respectively. This result suggests a heat-induced increase in surface charge density of the thylakoid membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES morpholinoethane sulfonic acid - MOPS 2-N-morpholinopropane sulfonic acid - TRICIN N-[tris(hydroxymethyl)-methyl] glycine     

Carbon-nitrogen requirements for the expression of nitrogenase activity in cultured Parasponia-Rhizobium strain ANU 289

Nutritional factors controlling derepression of nitrogenase activity in Parasponia-Rhizobium strain ANU 289 were studied in stationary and agitated liquid cultures. Altering type and/or concentrations of the constituents of the derepression medium in respect of carbon and nitrogen sources influenced both derepression kinetics as well as the maximal level of activity. Hexose sugars and disaccharides stimulated nitrogenase activity three to six-fold compared to pentose sugars. Activity was also modulated by combining sugars with some organic acids such as succinate, fumarate and pyruvate but not with others (e.g. -ketoglutarate, malate, malonate). Of the range of nitrogen sources tested, either casamino acids (at 0.05%, but not at 0.1%), glutamate, proline or to a lesser extent histidine (each at 5 mM N) supported significant derepression of nitrogenase activity. Notably glutamine, urea, alanine, ammonium sulfate, nitrate, nitrite (each at 5 mM N) and yeast extract (0.05%) failed to derepress or support nitrogenase activity. Ammonium (5 mM) abolished established nitrogenase activity of rapidly agitated cultures within 15 h after addition. This inhibitory effect was alleviated by the addition of methionine sulfoximime (10 mM). Thus, in view of strong glutamine effects, ammonium repression appears to be mediated by glutamine and not by ammonium itself.Abbreviations HEPES [4-(2-hydroxyethyl)-1-piperazine-ethane; sulfonic acid] - MOPS [3-(N-morpholino) propane sulphonic acid] - MSX Methionine sulfoximine     

Growth kinetics and nitrogenase induction inFrankia sp. HFPArI 3 grown in batch culture

Summary Kinetics of growth and nitrogenase induction inFrankia sp. Ar13 were studied in batch culture. Growth on defined medium with NH ₄ ⁺ as the N source displayed typical batch culture kinetics; however, a short stationary phase was followed by autolysis. Removal of NH ₄ ⁺ arrested growth and initiated vesicle differentiation. Vesicle numbers increased linearly and were paralleled by a rise in nitrogenase (acetylene reduction) activity. Nitrogenase activity (10 nM C₂H₄·mg protein^–1·min^–1) was sufficient to support growth on N₂ and protein levels rose in parallel with nitrogenase induction. Optimal conditions for vesicle and nitrogenase induction were investigated. Maximum rates of acetylene reduction were obtained with 5 to 10 mM K₂ HPO₄/KH₂PO₄, 0.1 mM CaCl₂ and MgSO₄. The optimum pH for acetylene reduction and respiration was around 6.7. The amount (5 to 10 g protein/ml) and stage (exponential) of growth of the ammonium-grown inoculum strongly influenced the subsequent development of nitrogenase activity. Propionate was the most effective carbon source tested for nitrogenase induction. Respiration in propionate-grown cells was stimulated by CO₂ and biotin, suggesting that propionate is metabolized via the propionyl CoA pathway.     

Ribulose bisphosphate carboxylase/oxygenase from Thiocapsa roseopersicina

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified 80-fold from malate-grown Thiocapsa roseopersicina by salting out the enzyme from the high-speed supernatant between 68–95% saturation with respect to (NH₄)₂SO₄, gelfiltration through Sephadex G-100, and DEAE-cellulose chromatography followed by sedimentation into a 14–34% glycerol gradient. The specific activity of enzyme for the carboxylase reaction was 2.45 mol RuBP-dependent CO₂ fixed/min · mg protein (at pH 8.0 and 30° C) and for the oxygenase reaction was 0.23 mol RuBP-dependent O₂ consumed/min · mg protein (at pH 8.6, and 25° C). The enzyme, which was ultracentrifugally homogeneous in the presence of 4 and 10% v/v glycerol, was stable for at least one year at-80° C in the presence of 10% glycerol. S_{20, w} values obtained in the presence of 4 and 10% glycerol were 19.3 and 16.2, respectively. The enzyme contained both large (53,000-daltons) and mixed small subunits (15,000- and 13,500-daltons).Borate-dependent inactivation of the enzyme by 2,3-butadione, which was greatly reduced in the presence of the product 3-phosphoglycerate, suggested that one or more arginines are at the active site.Abbreviations DTT dithiotreitol - RuBP d-ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate - TCA trichloroacetic acid - TEMBDG buffer (pH 8.0 at 25°C) containing 20 mM Tris, 1 mM disodium EDTA · 2 H₂O, 10 mM MgCl₂·6 H₂O, 50 mM NaHCO₃, 0.1 mM DTT and 10% glycerol (v/v)     

Some characteristics of symbiotic nitrogen fixation,yield, protein and oil accumulation in irrigated peanuts (Arachis hypogaea L.)

Summary The potential of peanuts for symbiotic nitrogen fixation is considerable and under optimal edaphic and climatic conditions it reached 222 kg N₂/ha, which was 58% of the nitrogen accumulated in the plants. The effect of the Rhizobium inoculation on crude protein accumulation in the yield (kg/ha) was 3–4 times greater than its effect on the yield of pods and hay. There was an inverse relationship between the protein and oil content in the kernels.Seasonal changes in nitrogenase activity in the nodules were determined by the acetylene reduction method during two growing seasons. Under favorable conditions the specific activity of the nitrogenase reached a very high level (up to 975 moles C₂H₂ g dry wt nod/h) and the total activity (moles C₂H₄/plant/h) was also high in spite of the relatively poor nodulation (weight and number). The high activity was drastically reduced (to 75 moles C₂H₄ g dry wt nod/h) due to exceptionally hot and dry weather, which occurred in the middle of the second half of the growing season. It appears that N₂-fixation (nitrogenase activity) is more sensitive to these unfavorable conditions, than is nodule growth. Maximum nitrogenase activity was observed during the podfilling stage; until 50–60 days after planting, nitrogenase activity was very low.