Isolation,distribution, and characterization of peach seedling nitrate reductase 1

Nitrate reductase (NR) was extracted from leaf, root, and stem tissue of ‘Lovell’ peach seedlings [Prunus persica (L.) Batsch] grown for 8 weeks in nutrient solution containing 15 mM nitrate. Enzyme activity of NR in leaf, stem, and root tissue was 10.20: 0.07: 0.04 nM N0₂/min/g tissue extracted, respectively. When seedlings wee transferred to nutrient solution containing 15 mM NH₄, NR activity was not detected after 72 hours. The enzyme was specific for NADH and had a pH optimum of 7.5. The K_m for NO₃ was 1.3 x 10–3 M and the rate of reaction remained linear for 45 min. Enzyme activity of leaf tissue was dependent on NO₃ concentration in the nutrient solution. At NO₃ concentrations of 15, 7.5, 1.5, and 0.15 mM, the NR activity was 22.8, 16.2, 13.8, and 2.2 nM NO₂/mg protein/hr.     

Kinetic behaviour of alkaline phosphatase desorbed from different clay minerals

The kinetic (K_m, V_max) of alkaline phosphatase (AP) desorbed from different Ca-homoionic clay minerals (montmorillonite, illite, and kaolinite) by extraction with Tris-Malate-Citrate buffer solution (pH 9.6) was studied in model experiments. After extraction (shaking for 15 min.) the K_m and V_max were measured in the extract, the remaining sediment and in the whole set-up. With kaolinite and illite, V_max of the desorbed AP was lower than that of the sediment. However, with montmorillonite, V_max of AP in the extract and whole system increased if compared to the control, but decreased in the sediment. The K_m of desorbed AP increased from 4.3 × 10^?3 (control) to 5.0 × 10^?3 M (illite), 5.4 × 10^?3M (kaolinite), and 5.5 × 10^?3M (montmorillonite). These values were lower than those obtained with the various sediments and whole experimental systems. An aberrant behaviour was recorded with the illite sorbed AP which showed an increase in affinity towards the substrate. Generally speaking, AP desorbed from clays may be reduced in its affinity towards the substrate p-nitrophenylphosphate by residual inhibitor and/or conformational change of the enzyme.     

The effect of soil pH and high urea concentrations on urease activity in soil

The rate of hydrolysis of urea in soil over the wide range of concentrations, up to 10 moles N per dm³ soil solution, found in fertilizer practice, was examined in Begbroke sandy loam adjusted to different pH values. On rewetting air-dry soil, urease activity increased rapidly, reached a maximum within the first 24 h and then decreased slowly to level off after about 4 days. Pretreatment of the soil with urea or ammonium had no effect on the urease activity. Urease activity increased with substrate concentration, reached an optimum value and then decreased with rising urea concentration. The results could be explained by substrate inhibition at higher urea concentrations, and the data are well described by a modified Michaelis-Menten equation involving three parameters, V_max, K_m and K_i where K_i is an inhibition constant. K_m decreased linearily with rise in pH whereas K_i increased slightly between pH 4.9 and 7.0 and steeply between 7.0 and 8.4. V_max increased with rise in pH, reached a maximum value at pH 6.0 and then declined at higher pHs. There was a further reaction, reaching a maximum rate at a urea concentration of about 0.2 molar N in the soil solution, that followed Michaelis-Menten kinetics. K_m for this high affinity reaction increased up to pH 7.2 and then decreased at higher pH values; V_max increased up to pH 6.8 and then decreased. The contribution of the high affinity reaction was small except at low concentrations of urea.     

Effect of different treatments with crop residues on soil phosphatase activity

Summary Phosphomonoesterase (both acid and alkaline) and phosphodiesterase activity was either activated or inhibited in a soil treated with different crop residues. Phosphotriesterase activity remained unaffected. The kinetic parameters (V _max and K _m) of treated soil samples were modified in the same way: Increases or decreases in the V _max values corresponded to increases or decreases in the K _m values. The V _max values, rather than the K _m values, were found to have a predominant effect on phosphatase activity, thus indicating a fundamental role for the enzyme concentration. A positive and generally significant correlation was found between the activity of each phosphatase, which suggests an unspecific source of these enzymes. The values of the determination coefficients (R ² × 100) show that a low percentage of the variability may be ascribed to interactions among phosphatase activities.     

喀斯特石漠化山区苔藓植物水分吸收特征

[目的]探究贵阳市花溪区石漠化地区5种优势种黑扭口藓(Barbula nigrescens)、美灰藓(Eurohypnum leptothallum)、卷叶湿地藓(Hyophila involuta)、小牛舌藓全缘亚种(Anomondon minor subsp.integerrimus)、北方紫萼藓(Grimmia decipiens)的水分吸收特征,为喀斯特石漠化地区利用苔藓植物开展水土保持工作提供理论支撑。[方法]采集样品120份,运用经典形态分类法进行鉴定;测定其生物量、饱和吸水率、吸水量、最大吸水速率(Vmax)、吸水速率常数(Km)和叶片展开时间。[结果](1)5种苔藓植物间的生物量、吸水量和饱和吸水率差异较大,最大吸水速率差异小;生物量为10.36~114.51g/m2;饱和吸水率为675.43 6%~1 125.41%;吸水量为98.21~766.13g/m2;Vmax为35.59~51.28g/(g·min);Km为69.97~101.99g;叶片展开时间为35.9~86.1s。(2)生物量和吸水量呈正极显著相关;吸水量和盖度、Vmax和Km呈正相关;Km和叶片展开时间呈负相关。[结论]在喀斯特石漠化地区干旱缺水的环境条件下,苔藓以独特的水分吸收和利用方式适应这种恶劣的环境。石生苔藓为适应该地区的先锋植物。     

Purification and Characterization of a Hexose Oxidase with Excellent Strengthening Effects in Bread

Hexose oxidase (EC 1.1.3.5) (HOX) was purified 51-fold from the red algae Chondrus crispus, by several chromatography methods, including hydrophobic interaction, chelating Sepharose, anion exchange, gel filtration, and chromatofocusing. Purified HOX was subjected to native PAGE and activity staining with nitroblue tetrazolium. For HOX electroeluted out of the gel and digested with endoproteinase Lys-C, the internal peptide sequence determined was: D-P-G-Y-I-V-I-D-V-N-A-G-T-(V or P)-D-K-P-D-P-X. The molecular mass, determined by gel filtration, was 126 kDa, versus 65 kDa determined by SDS-PAGE. The pI was determined to 4.64 and 4.79 as a double band on an isoelectrofocusing gel. K_m was determined to 2.7 mM for D-glucose, 3.6 mM for D-galactose, 20.2 mM for cellobiose, 43.7 mM for maltose, 90.3 mM for lactose, 102 mM for xylose, and 531 mM for arabinose. The oxidation of thiol groups in gluten was determined by using Ellman's reagent: 5,5′-dithiobis (2-nitrobenzoic acid). The effect of HOX was compared to that of glucose oxidase. Both enzymes caused a dose-responsive reduction in the free thiol groups. Extensigraph measurements and baking tests confirmed that HOX caused increased dough strength and increased bread volume more efficiently than glucose oxidase used in the same dosage.     

Adsorption,activity, and kinetics of acid phosphatase as influenced by selected oxides and clay minerals

Abstrac

The effects of 3 oxides (Fe, Al, and Mn oxides) and 3 clay minerals (kaolin, montmorillonite, and allophane) on the adsorption and subsequent kinetic properties of acid phosphatase were compared. The amount of enzyme adsorbed by the oxides and clay minerals followed the order: montmorillonite ? kaolin > Mn oxide > Fe oxide > Al oxide ? allophane. The adsorption isotherms of the enzyme on the oxides and clay minerals, except for montmorillonite and allophane, fitted the Langmuir equation. The activity of the enzyme immobilized by the inorganic components studied was in the order of allophane > kaolin > Fe oxide > montmorillonite > Al oxide ≒ Mn oxide. Compared to the free enzyme, the V _max, K_m, and V _max / K _m values of the immobilized enzyme decreased, increased, and decreased, respectively. Among the oxides or clay minerals, the higher the ability of the inorganic components to adsorb the enzyme, the lower the value of the V _max / K _m ratio of the immobilized enzyme. These findings suggest that the catalytic efficiency of the enzyme complexes formed is determined by the adsorbability of the inorganic components for the enzyme.     

Large-Scale Purification and Characterization of Barley Limit Dextrinase,a Member of the α-Amylase Structural Family

Homogeneous barley limit dextrinase (LD) was isolated on a large scale in a yield of 9 mg/kg of 10-day germinated green malt. This represents a 9,400-fold purification and 29% recovery of the activity in a flour extract in 0.2M NaOAc (pH 5.0) containing 5 mM ascorbic acid. The purification protocol consists of precipitation from the extract at 20–70% saturated ammonium sulfate (AMS), followed by diethylaminoethyl (DEAE) 650S Fractogel anion-exchange chromatography, and affinity chromatography on β-cyclodextrin-Sepharose in the presence of 2M AMS. LD was eluted by 7 mMβ-cyclodextrin and contains a single polypeptide chain of 105 kDa (SDS-PAGE) and pI 4.3. Sequence analysis of tryptic fragments, prepared from 2-vinylpyridinylated LD and purified by RP-HPLC, identified short motifs recognized in β-strand 2, 3, and 5 characteristic of a catalytic (β/α)₈-barrel domain of the α-amylase family of amylolytic enzymes. Barley LD has ≈50 and 85% sequence identity to bacterial pullulanases and rice starch debranching enzyme, respectively. By using ¹H-NMR spectroscopy, LD hydrolyzes specifically α-1,6-glucosidic linkages in pullulan and a branched oligodextrin, 6²-O-α-maltotriosyl-maltotriose, with retention of the α-anomeric configuration. β-Cyclodextrin competitively inhibits the LD activity with K_i of 40 μM, while K_i is 1.9 mM and 2.4 mM for α-cyclodextrin and γ-cyclodextrin, respectively.     

Iron and zinc absorption characteristics and copper inhibitions in cucurbitaceae

Iron and Zn absorption, interactions, and Cu inhibitions were characterized in cucumber (Cucumis sativus L.), watermelon (Citrullus lanatus Thunb.), and pumpkin (Cucurbita moschata Poir.) by kinetic parameters V_max and K_m. Influx and V_max values for Fe and Zn absorption decreased in each species as plant age increased. For the Michaelis constant, K_m, Fe values in cucumber and watermelon and Zn values in watermelon and pumpkin were relatively unchanged with increased plant age. K_m values for Zn absorption in cucumber and Fe absorption in pumpkin decreased as plant age increased. Among species, watermelon appeared to have a particularly effective uptake mechanism for Zn at low solution concentrations. Non‐competitive inhibition of Zn absorption by Fe (20, 50 uM) was indicated in each species. Iron uptake in pumpkin was inhibited non‐competitively by Zn (5, 10 uM), however no significant effects of Zn on Fe absorption were evident in either watermelon or cucumber. Copper (0.5, 1, 5 uM) inhibited uptake of Fe non‐competitively and Zn competitively in each species.     

Kinetic parameters of dehydrogenase in the assessment of the response of soil to vermicompost and inorganic fertilisers

 Kinetic parameters (V _max and K _m) of dehydrogenase activity were determined in order to assess the metabolic response of a soil about 1 year after organic and mineral treatments. The soil was planted with maize (Zea mays) and treated with the following fertilisers: organic (vermicompost; VC), mineral (ammonium phosphate and urea), and an organo-mineral mixture. V _max, which represents a measurement of the quantity of enzyme, markedly increased in organic and organo-mineral treatments, indicating that the addition of organic matter caused an increase in dehydrogenase in the active microbial biomass. K _m, representing enzyme-substrate affinity and/or different sources of the enzymes, was similar in VC-treated soil and control soil, while it doubled in organo-mineral and mineral treatments. These results suggest that the use of VC did not alter the enzyme-substrate affinity, while mineral fertiliser reduced this affinity or changed the composition and activity of soil microbiota. A positive correlation was found between V _max, the metabolic index (dehydrogenase/water-soluble carbon ratio), and the soil organic matter content. The kinetic constants of dehydrogenase activity and the metabolic index may be considered valid parameters to monitor the evolution of microbiological activity in soil. Received: 4 February 2000     

连续6年施用有机肥处理对水稻土磷酸单酯酶活性、动力学和热力学特性的影响

Soil phosphomonoesterase plays a critical role in controlling phosphorus(P) cycling for crop nutrition,especially in P-deficient soils.A 6-year field experiment was conducted to evaluate soil phosphomonoesterase activities,kinetics and thermodynamics during rice growth stages after consistent swine manure application,to understand the impacts of swine manure amendment rates on soil chemical and enzymatic properties,and to investigate the correlations between soil enzymatic and chemical variables.The experiment was set out in a randomized complete block design with three replicates and five treatments including three swine manure rates(26,39,and 52 kg P ha~(-1),representing low,middle,and high application rates,respectively) and two controls(no-fertilizer and superphosphate at 26 kg P ha~(-1)).The results indicated that the grain yield and soil chemical properties were significantly improved with the application of P-based swine manure from 0 to 39 kg P ha~(-1);however,the differences between the 39(M_(39)) and 52 kg P ha~(-1) treatments(M_(52)) were not significant.The enzymatic property analysis indicated that acid phosphomonoesterase was the predominant phosphomonoesterase in the tested soil.The M_(39) and M_(52) treatments had relatively high initial velocity(V_0),maximal velocity(V_(max)),and activation grade(lgN_a) but low Michaelis constant(K_m),temperature coefficient(Q_(10)),activation energy(E_a),and activation enthalpy(ΔH),implying that the M_(39) and M_(52) treatments could stimulate the enzyme-catalyzed reactions more easily than all other treatments.The correlation analysis showed that the distribution of soil phosphomonoesterase activities mainly followed the distributions of total C and total N.Based on these results,39 kg P ha~(-1) could be recommended as the most appropriate rate of swine manure amendment.     

Biodegradation kinetics of monosaccharides and their contribution to basal respiration in tropical forest soils

Low molecular weight (LMW) organic compounds in soil solution are easily biodegradable and could fuel respiration by soil microorganisms. Our main aim was to study the mineralization kinetics of monosaccharides using ¹⁴C-radiolabelled glucose. Based on these data and the soil solution concentrations of monosaccharides, we evaluated the contribution of monosaccharides to basal respiration for a variety of tropical forest soils. Further, the factors controlling the mineralization kinetics of monosaccharides were examined by comparing tropical and temperate forest soils. Monosaccharides comprised on average 5.2 to 47.7% of dissolved organic carbon in soil solution. Their kinetic parameters (V _max and K_M ), which were described by a single Michaelis-Menten equation, varied widely from 11 to 152?nmol?g^?1?h^?1 and 198 to 1294?µmol?L^?1 for tropical soils, and from 182 to 400?nmol?g^?1?h^?1 and 1277 to 3150?µmol?L^?1 for temperate soils, respectively. The values of V _max increased with increasing microbial biomass-C in tropical and temperate soils, while the K_M values had no correlations with soil biological or physicochemical properties. The positive correlation between V _max values and microbial biomass-C indicates that microbial biomass-C is an essential factor to regulate the V _max values in tropical and temperate forest soils. The biodegradation kinetics of monosaccharides indicate that the microbial capacity of monosaccharide mineralization far exceeds its rate at soil solution concentration. Monosaccharides in soil solution are rapidly mineralized, and their mean residence times in this study were very short (0.4–1.9?h) in tropical forests. The rates of monosaccharide mineralization at actual soil solution concentrations made up 22–118% of basal respiration. Probably because of the rapid and continuous production and consumption of monosaccharides, monosaccharide mineralization is shown to be a dominant fraction of basal respiration in tropical forest soils, as well as in temperate and boreal forest soils.     

GROWTH AND POTASSIUM TRANSPORT IN COMMON AND DURUM WHEAT AS AFFECTED BY ALUMINUM AND NITRITE STRESS

Aluminum (Al) toxicity has been identified as one of the most important factors limiting plant growth in acid soil. Besides Al, nitrite (NO₂ ^?) may also be a significant stress factor in an acid environment. The objective of this study was to examine the effects of Al and NO₂ ^? stress on the growth and potassium (K⁺) uptake of roots and their transport toward the shoots of an Al-resistant common wheat (Triticum aestivum L. cv. Jubilejnaja 50) and an Al-sensitive durum wheat (T. durum Desf. cv. GK Betadur) grown in 0.5 mM CaSO₄ solution at pH 4.1 or 6.5. Root elongation of durum wheat was inhibited with 30% at 10 μM AlCl₃ treatment, while this low Al-concentration did not show a significant effect on root growth of common wheat. In all cases shoot growth was not influenced under low-salt conditions by 10 μ M AlCl₃, but exposure to 100 μM KNO₂ (alone or in combination with Al) had a definite stimulatory effect on growth. Aluminum was found to stimulate the K⁺(⁸⁶Rb) influx in short-term (6 h) experiments, but to inhibit it in long-term (3 days) experiments. This treatment was thought to damage the plasma membrane. When 10 μM 2,4-dinitrophenol was present in the uptake solution the Al-stimulated K⁺ uptake stopped even in short-term experiments. In the case of nitrite and nitrite + Al treatment combinations, however, a striking inhibition was observed in the K⁺(⁸⁶Rb) influx and the K⁺ concentration of the roots and shoots of both species.     

Synthesis of 15N-labelled microbial biomass in soil in situ and extraction of biomass N

Summary A Pakistani soil (Hafizabad silt loam) was incubated at 30°C with varying levels of ¹⁵N-labelled ammonium sulphate and glucose (C/N ratio of 30 at each addition rate) in order to generate different insitu levels of ¹⁵N-labelled microbial biomass. At a stage when all of the applied ¹⁵N was in organic forms, as biomass and products, the soil samples were analysed for biomass N by the chloroform (CHCl₃) fumigation-extraction method, which involves exposure of the soil to CHCl₃ vapour for 24 h followed by extraction with 500 mM K₂SO₄. A correction is made for inorganic and organic N in 500 mM K₂SO₄ extracts of the unfumigated soil. Results obtained using this approach were compared with the amounts of immobilized ¹⁵N extracted by 500 mM K₂SO₄ containing different amounts of CHCl₃. The extraction time varied from 0.5 to 4 h.The amount of N extracted ranged from 27 to 270 g g^–1, the minimum occurring at the lowest (67 g g^–1) and the maximum at the highest (333 g g^–1) N-addition rate. Extractability of biomass ¹⁵N ranged from 25% at the lowest N-addition rate to 65%a for the highest rate and increased consistently with an increase in the amount of ¹⁵N and glucose added. The amounts of both soil N and immobilized ¹⁵N extracted with 500 mM K₂SO₄ containing CHCl₃ increased with an increase in extraction time and in concentration of CHCl₃. The chloroform fumigation-extraction method gives low estimates for biomass N because some of the organic N in K₂SO₄ extracts of unfumigated soil is derived from biomass.     

A comparison of methods for estimating phosphorus uptake kinetics under steady‐state conditions 1

Michaelis‐Menten kinetic parameters (I_max and K_M) are useful for describing nutrient uptake by plants. This paper compares two methods for estimating the kinetics of P uptake. Both methods employed a steady‐state hydroponic system to measure P uptake by wheat (Triticum aestivum L.) seedlings. In one method, uptake was measured from two P concentrations in nutrient solution, with I_max and K_M estimated by direct linear plot (DLP). In an alternate, multiple concentration (MC) method, uptake was measured from five P concentrations, and kinetic parameters were estimated by either nonlinear regression or the Hanes plot. The I_max and K_M, estimates obtained by the DLP method were compared to those obtained by the MC method. The MC method offered practical advantages. Unlike the DLP, it allowed estimation of the external P concentration at which net influx = 0 (C_min), and did not require a priori estimates of K_M and C_min. The MC method provided more precise median parameter estimates as indicated by smaller nonparametric confidence intervals. Using the median C_min value of 1.9 μM, the best estimates of I_max and K_M (and 96% confidence intervals) derived by nonlinear regression were 2.2 (1.6 to 2.8) nmol P g^‐1s^‐1, and 11 (10.6 to 12.9) μM, respectively.     

Influence of natural and synthetic humic substances on the activity of urease

The activity of a purified urease, obtained from Bacillus pasteurii, was inhibited by humic and fulvic acids obtained from an agricultural soil. Enzyme kinetic studies showed that the humic substances affected the affinity of the enzyme for its substrate (K_m) and the maximum velocity of the reaction (V_max). The V_max was inhibited to the same extent by both humic (HA) and fulvic (FA) acids, the precise effect depending on the pH and concentration of humic substance. At pH 4.0, HA concentrations of 25 pg cm^?3 and 10 μg cm^?3 inhibited the V_max by 38.5% and 20% respectively. HA and FA had similar effects on the K_m but in this case the lowering of the affinity of the enzyme for its substrate was not concentration dependent in the range 0–25 μg cm^?3 of humic substance. Typically, the affinity was decreased from a K_M of 50 mM in the control to 67 mM in the presence of HA and FA. The effects were not due primarily to the ash or N contents of the humic substances because de-ashed humic acid and synthetic model humic (made from catechol, guaiacol, pyrogallol, resorcinol and protocatechuic acid) and fulvic acid (made from polymaleic acid), containing virtually no ash or N, were equally as effective. The effect was not related to the phenolic monomers which, before polymerization, had no effect on urease activity.     

Ermittlung kinetischer Parameter der sauren Phosphatasen intakter Zuckerrübenwurzeln bei variierter Phosphaternährung

Determination of kinetic parameters of acid phosphatases in intact sugar beet roots of variable phosphorus nutrition Organically bound phosphorus has to be hydrolysed before its P can be taken up by plants. Both microbes and plant roots possess phosphatases, which could be of importance especially in soils with low concentrations of inorganic phosphorus in the soil solution. This could be the reason why nutrient uptake models underestimate the P-uptake by plants when P-mobilization by the phosphatases of roots is not taken into consideration. Therefore the activity of acid phosphatases (P_ase) was determined to answer the following questions: 1) To which extent does the root bound acid phosphatase (P_ase) follow Michaelis-Menten kinetics? 2) By which of the four linear transformations of the Michaelis-Menten equation (Lineweaver/Burk, Hanes, Eadie/Hofstee, Eisenthal/Cornish-Bowden) can plausible values of V_max and K_m be determined? 3) Which effect has the P nutrition of the plant on these kinetic parameters? Sugar beet plants were grown in full nutrient solution containing 1 and 100 μM P respectively. The P_ase activity of the intact roots was measured at pH 5 using p-nitrophenylphosphate (25—15000 μM p-NPP). V_max values were calculated per m root length. Acid phosphatase activity principally followed Michaelis-Menten kinetics. Transformations and calculations of V_max and K_m after Eadie/Hofstee and Eisenthal/Cornish-Bowden suggested the existence of at least two enzyme systems (P_{ase 1}, P_{ase 2}). The following kinetic parameters were found: P_{ase 1}: P deficient plants: V_max: 43—45 nmol m^—1 min^—1, K_m: 31—37 μM NPP; P sufficient plants: V_max: 7 nmol m^—1 min^—1, K_m: 47—53 μM NPP. P_{ase 2}: P deficient plants: V_max: 230—293 nmol m^—1 min^—1, K_m: 1579—3845 μM NPP; P sufficient plants: V_max: 123—171 nmol m^—1 min^—1, K_m: 3027—7000 μM NPP. Thus plants with sufficient P nutrition have a lower affinity to P_org and a lower hydrolysis of P_org. For P nutrition of crops P_{ase 1} might be the most important enzyme.     

A Practical Spectrophotometric Approach for the Determination of Lipoxygenase Activity of Durum Wheat

In this study, a practical spectrophotometric approach was used to determine the hydroperoxidation activity of durum wheat lipoxygenase (LOX). As stated in the related literature, the buffered linoleic acid solution used as the reaction medium is not optically clear enough at neutral and lower pH values due to its limited solubility. In our study, the optical clarity was obtained by the formation of sodium-salt of unreacted linoleic acid just before absorbance measurement. The durum wheat LOX was characterized in terms of pH and temperature optima as well as kinetic parameters. The maximum linoleic acid hydroperoxidation activities were determined at pH 5.0 and 6.5 and at 40°C This result can be considered as evidence for the presence of at least two LOX isoforms with respective optima at pH 5.0 and 6.5 in the crude durum wheat extract. The Michaelis constant (K_m) and maximum hydroperoxidation) activity rate (V_max) of durum wheat LOX for linoleic acid were estimated) to be 0.131 ± 0.019 mM and 42.37 ± 3.32 units/mg of protein/min, respectively. The method seems to be useful for the determination of LOX activity in durum wheat and its milling fractions.     

Influence of plant growth regulators on some enzymes of nitrogen assimilation in mustard seedlings

Effect of plant growth regulators (PGR) viz. gibberellic acid (GA), kinetin (KN), and abscissic acid (ABA) were investigated on growth and activities of nitrate reductase (NR) and glutamine synthetase (GS) in mustard (Brassica juncea) seedlings. All the PGRs tested promoted in vivo NR activity in cotyledons, but the magnitude differed with different treatments. Cytosolic GS in root and hypocotyl was promoted by GA treatment and inhibited by ABA and KN treatments although the latter showed slight promotion initially in hypocotyl; the trend was not clear in cotyledons. Determination of K_m value of GS extracted from 96‐h‐old cotyledons recorded lower K_m value in GA treatment (2.5 mM), while it increased in ABA treatment (4.35 mM), There was little change in K_m value in KN (3.03 mM) treatment. The kinetics of GS enzyme in cotyledons of different treatments showed marked variation in V_max . Both GA and ABA treatments inhibited GS activity while no significant effect by KN treatment was observed. It is argued that GA treatment inhibits chloroplastic GS (an enzyme which has higher K_m value), while ABA‐induced inhibition may not be specific to cytosolic or chloroplastic isoforms. Kinetin treatment was ineffective in promoting or inhibiting GS activity in cotyledons. The above conclusion is further supported by chloroplastic pigment data where inhibition is recorded in all the PGRs tested.     

Characterization of free and adsorbed phosphatases in soils

Summary The kinetic and thermodynamic parameters of soil alkaline phosphatases, extracted humus-enzyme complexes, and water extracts were evaluated. The isolation procedure for humus-enzyme complexes involves exhaustive extraction of the soil with the chelating resin Chelex 100. The K _m values for alkaline phosphatases in the resin extracts of two soils were 0.881 and 2.236 mM, respectively, and were greater than those of the water extracts (0.320 and 0.527 mM) and the soil suspensions (0.445 and 0.652 mM). The v _max values varied considerably, indicating the fundamental role of the enzyme concentration in the fractions and soils. The temperature of inactivation in the water extracts was 30°C, being 10° and 20°C lower than the temperature needed to inactivate alkaline phosphatase in humus-enzyme complexes and in soils, respectively. The results indicated that only a small portion of the enzyme activity was in a soluble state and that this fraction was characterized by a low thermal stability. The sorption of alkaline phosphatases to humic substances gives them additional thermal stability and leads to a partial inhibition of the enzyme.Dedicated to the late Prof. Dr. W. Kühnelt