Effects of pH on ammonium uptake by Typha latifolia L.

The effects of solution pH on NH₄⁺ uptake kinetics and net H⁺ extrusion by Typha latifolia L. were studied during short-term (days) and long-term (weeks) exposure to pH in the range of pH 3.5–8.0. The NH₄⁺ uptake kinetics were estimated from depletion curves using a modified Michaelis-Menten model. T. latifolia was able to grow in solution culture with NH₄⁺ as the sole N source and to withstand a low medium pH for short periods (days). With prolonged exposure (weeks) to pH 3.5, however, the plants showed severe symptoms of stress and stopped growing. The solution pH affected NH₄⁺ uptake kinetics. The affinity for NH₄⁺, as quantified by the half saturation constant (K_1/2) and C_min (the NH₄⁺ concentration at which uptake ceases), decreased with pH. K_1/2 was increased from 7.1 to 19.2 mmol m^?3 and C_min from 2.0 to 5.7 mmol m^?3 by lowering the pH in steps from 8.0 to 3.5. V_max was, however, largely unaffected by pH (～22 μmol h^?1 g^?1 root dry weight). Under prolonged exposure to constant pH, growth rates were highest at PH 5.0 and 6.5. At pH 8.0 growth was slightly depressed and at pH 3.5 growth completely stopped. NH₄⁺ uptake kinetics were similar at pH 5.0, 6.5 and 8.0 whereas at pH 3.5 NH₄⁺ uptake almost completely stopped. The ratio between net H⁺ extrusion and NH₄⁺ uptake decreased significantly at low pH. The adverse effects of low pH on NH₄⁺ uptake kinetics are probably a consequence of a reduced H⁺-ATPase activity and/or an increased re-entry of H⁺ at low pH, and the associated decrease in the electrochemical gradient across the plasma membranes of the root cells.     

Kinetics of Nitrosomonas europaea at extreme substrate,product and salt concentrations

Summary Nitrification of ammonia in concentrated waste streams is gaining a lot of attention nowadays. Nitrosomonas europaea is the predominant ammonia-oxidizing species in these environments. Prediction of the behaviour of a pure culture of N. europaea (ATCC 19718) under conditions prevailing in concentrated waste streams was the aim of this study. The initial oxygen consumption rate of a concentrated cell suspension was used as a rapid assay to measure the effects on N. europaea under various conditions. Several relationships, based on Michaelis-Menten kinetics, were derived. They describe the behaviour of N. europaea at substrate (NH₄ ⁺), product (NO₂ ^– and K⁺, Na⁺, SO _inf4 ^sup2– , NO₃ ^–, Cl^–) concentrations up to 500 mol/m³ and pHs ranging from 6.5 to 8.5. High concentrations of ions inhibited N. europaea but specific substrate inhibition was not observed. Product inhibition was strongly pH-dependent and severe inhibition was found at pH 6.5. Correspondence to: J. H. Hunik     

Chitin synthetase from the yeast and mycelial phases of Blastomyces dermatitidis

Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 ° C; pH optimum was 7–7.5; Mg⁺² optimum was 5–10 mM.The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment.     

NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings

Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21-day-old spruce (Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD⁺. Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD⁺-IDH activity was about 2-fold higher than NADP⁺-IDH activity. The NAD⁺-IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis-Menten kinetics for NAD⁺ and Mg²⁺. The NADP⁺-IDH, in contrast, displayed lower K_m values. For NAD⁺-IDH the pH optimum was at 7.4, whereas NADP⁺-IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD⁺-IDH was more thermolabile. Adenine nucleotides and 2-oxoglutarate were found to inhibit NAD(P)⁺-IDH activities only at high concentrations.     

The kinetics of NH4 uptake by Ceratophyllum

The relationship between growth of aquatic plants and their nutrient supply is poorly understood, because of the lack of suitable models which could be tested in the field. The purpose of this research was to learn if the Michaelis-Menten expression describes the relationship between the uptake of NH₄ by Ceratophyllum and the concentration of NH₄ in solution.A hyperbola results when uptake of NH₄ by Ceratophyllum (v) is plotted against the concentration of NH₄ in solution (S). The relationship is not described well by the Michaelis-Menten expression. Linearily between v and S at concentrations less than 7 mg NH₄-N(1)-¹, suggests that a model for first order saturation kinetics might be appropriate. However, uptake is depressed by one half at low temperatures and some of the uptake is apparently related to processes which are not completely passive. The ecological usefulness of the Michaelis-Menten constants derived for Ceratophyllum is discussed.     

Phosphatidate phosphatase from avocado (Persea americana) – purification, substrate specificity and possible metabolic implications for the Kennedy pathway and cell signalling in plants

Phosphatidate phosphatase (PAP; EC 3.1.3.4) is a pivotal enzyme in plant lipid metabolism positioned at a major branchpoint between the biosyntheses of phospholipids and triacylglycerols. The enzyme has been purified 7000-fold from the microsomes of maturing Avocado fruit. The enzyme has a subunit molecular mass, as determined on SDS-PAGE, of 49 kDa, and gel filtration studies revealed that it is monomeric. The enzyme was examined for the ability to hydrolyse sn-1,2-dioleoylglycerol-3-phosphate (PA), sn-1-oleoylglycerol-3-phosphate (LPA), sn-2-oleoylglycerol-3-phosphate (sn-2-LPA), and ceramide-1-phosphate. All substrates were used, but the apparent V_max values for ceramide-1-phosphate and sn-2-LPA were considerably lower than for PA or LPA. A kinetic analysis of the purified enzyme was conducted using PA and LPA in Triton X-100 mixed micelles according to the surface dilution kinetic model. The V_max and interfacial Michaelis constant (Km^B) were calculated for PA as 47 μmol min^–1 mg^–1 and 1.49 mol%, respectively. LPA was found to be a better substrate with a V_max and Km^B of 100 μmol min^–1 mg^–1 and 1.06 mol%, respectively. A detailed kinetic analysis of the effect of LPA on PA hydrolysis revealed LPA to be a potent competitive inhibitor of the reaction, with a K_i of 1.4 mol%. This possibly indicates that strict metabolic channelling is in operation in the Kennedy Pathway, with very low steady state concentrations of lyso-phosphatidate with respect to phosphatidate in vivo.     

Reproduction in two submersed macrophytes declines progressively at low pH

• 1 Greenhouse experiments tested the effect of pH 5 v pH 7.5 on reproductive success for the freshwater macrophytes Najas flexilis, an annual, and Vallisneria americana, which perennates as a tuber.
• 2 Seed production by the small Najas plants grown at pH 5 averaged 0.25 seeds/plant, in contrast to 95.5 seeds/plant at pH 7.5. At low pH, Vallisneria grown from seeds produced no flowers and too few tubers to replace themselves, so that sexual reproduction failed nearly completely for both species.
• 3 Vallisneria grown from tubers produced 97% less total tuber mass at pH 5 (0.4 v 14.9 g), the compounded result of producing, on average, 89% fewer and 82% smaller tubers. The smaller tubers developed at pH 5 were less likely to overwinter in the field, and those surviving tubers subsequently grew into smaller plants.
• 4 These findings generated the hypothesis of a closing spiral: growth at low pH (and relatively low CO₂ concentrations) results in small plants that produce a smaller number of progressively smaller tubers each autumn, which in turn develop into progressively smaller plants each summer. This hypothesis was supported by field transplant experiments in two acidic Adirondack Mountain (NY) lakes.

     

Catabolism of d-fructose and d-ribose by Pseudomonas doudoroffii

1. The 1-P-fructokinase (1-PFK) and 6-P-fructokinase (6-PFK) from Pseudomonas doudoroffii were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. The pH optima of these enzymes were 9.0 and 8.5, respectively.
2. When the concentrations of the substrates of the 1-PFK reaction were varied, Michaelis-Menten kinetics were observed. The Kms for d-fructose-1-P (F-1-P) and ATP were 3.03×10^-4 M and 3.39×10^-4 M, respectively. Variation of MgCl₂ at fixed concentrations of F-1-P and ATP resulted in sigmoidal kinetics; about 10 mM MgCl₂ was necessary for maximal activity. Activity of 1-PFK was inhibited when the ratio of ATP: Mg⁺⁺ was higher than 0.5, suggesting that ATP: 2Mg⁺⁺ was the substrate and that free ATP was inhibitory. Although an absolute requirement for K⁺ or NH ₊ ⁴ could not be demonstrated, these cations stimulated the rate of the reaction. Activity of 1-PFK was not significantly affected by 3 mM AMP, cyclic-AMP, P_i, d-fructose-6-P (F-6-P), ADP, P-enolpyruvate (PEP), pyruvate, citrate, or l-glutamate.
3. Sigmoidal kinetics were observed for 6-PFK when the concentration of F-6-P was increased and the level of ATP was kept constant. Activity of 6-PFK was increased by ADP, inhibited by PEP, and unaffected by 3 mM AMP, cyclic-AMP, P_i, F-1-P, pyruvate, or citrate.

     

Glutamate dehydrogenase of lupin nodules: Purification and properties

Glutamate dehydrogenase, GDH (l-glutamate: NAD⁺ oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45 000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD⁺ for the deamination reaction. Pyridoxal-5′-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD⁺. When NAD⁺ was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD⁺.     

Glutamine uptake by a sodium-dependent secondary transport system inCorynebacterium glutamicum

Corynebacterium glutamicum took up glutamine by a sodium-dependent secondary transport system. Both the membrane potential and the sodium gradient were driving forces. Glutamine uptake showed Michaelis-Menten kinetics, with aK _m of 36 μM and aV _max of 12.5 nmol min⁻¹ (mg dry weight)⁻¹ at pH 7. Despite a pH optimum in the alkaline range around pH 9, it was shown that uncharged glutamine is the transported species. The affinity for the cotransported sodium was relatively low; an apparentK _m of 1.4 mM was determined. Among various substrates tested, only asparagine, when added in 50-fold excess, led to an inhibition of glutamine transport. It was concluded that glutamine uptake occurs via a specific transport system in symport with at least one sodium ion.     

Isolation and partial characterization of a cyclic GMP-dependent cyclic GMP-specific phosphodiesterase from Dictyostelium discoideum

The cellular slime mold, Dictyostelium discoideum, contains at least two classes of phosphodiesterase activity. One class of enzymes hydrolyses cyclic AMP (cAMP) and cyclic GMP (cGMP) with approximately equal rates. Another enzyme, which is less than 5% of the total activity, specifically hydrolyses cGMP. The cGMP-specific enzyme does not bind to a Con A-Sepharose column, while all the cAMP-hydrolyzing activities are retarded by this column. The cGMP-specific enzyme is activated by low cGMP concentrations (10^?8-10^?6 M); the enzyme has normal Michaelis-Menten kinetics at high substrate concentrations with a K_m of about 3–6 μM. The cGMP-binding sites for activation and for catalysis show different cyclic nucleotide specificity, but they are probably located on one protein with a molecular weight of about 70 000. The enzyme is stable only under specific conditions, and the activation property of the enzyme is lost relatively easy. Irreversible modifications occur at temperatures below 0° and above 30°C, and at pH below 6.0. Several other conditions such as high ion concentrations, temperatures just above 0°C and pH above 8.0 lead to reversibel modifications of enzyme activity.     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

Kinetics of ionic transport across frog skin: Two concentration-dependent processes

Summary Sodium and chloride influxes across the nonshort-circuited isolated skin ofRana esculenta were measured at widely varying external ionic concentrations.The curve describing sodium transport has two Michaelis-Menten components linked at an inflection point occurring at an external sodium concentration of about 7 meq. Chloride transport can also be represented by two saturating components. A possible explanation of these kinetics is discussed.At sodium concentrations lower than 4 meq it is possible to define a component of the sodium transport mechanism as having a high affinity for sodium and which is independent of the nature of the external anion. A high affinity for chloride of the chloride transport system functioning at low external concentrations is also found but is significantly different from that of sodium. These systems show the physiological characteristics of the countertransports (Na _ext ⁺ /H _int ⁺ ; Cl _ext ^– /HCO _3int ^– ) functioning at low external concentrations.At external concentrations higher than 4 meq a low affinity transporting system in which chloride and sodium are linked superimpose on the high affinity components.The physiological significance of these results is discussed.     

Invasive submerged freshwater macrophytes are more plastic in their response to light intensity than to the availability of free CO2 in air‐equilibrated water

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Purification and characterization of a <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase produced by <Emphasis Type="Italic">Talaromyces emersonii</Emphasis> during growth on algal fucoidan

A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (K_m value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH 5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg²⁺, Co²⁺ and Fe³⁺.     

Radial oxygen loss by the cushion plant Eriocaulon schimperi prevents methane emissions from an East‐African mountain mire

• Groundwater‐fed fens are known sources of methane (CH₄) emissions to the atmosphere, and these are known to be mediated by the vegetation. In a fen located in the Bale Mountains, Ethiopia, we assessed the effects of a cushion plant (Eriocaulon schimperi) and a sedge (Carex monostachya) on rhizosphere biogeochemistry.
• Methane and CO₂ concentrations and pH were measured in pore‐water at different depths in the profile. Redox potentials and NaCl‐extractable element concentrations were analysed in soil samples from sites dominated by either E. schimperii or C. monostachya. Nutrient and element concentration were analysed in plant tissues.
• At Carex‐dominated sites, CH₄ concentrations increased from 70 μmol·l^?1 at a depth of 10 cm to 130 μmol·l^?1 at a depth of 100 cm. CH₄ concentrations at Eriocaulon‐dominated sites were almost zero (<1 μmol·l^?1) to a depth of 100 cm. Simultaneously, soil redox potentials and CO₂ concentrations were higher at Eriocaulon‐dominated sites, indicating a low potential for CH₄ production and a high potential for CH₄ oxidation.
• Eriocaulon schimperi displayed a root investment strategy to cope with the harsh environment, similar to the cushion plant Astelia pumila in Patagonian bogs. This strategy is characterised by high root/shoot ratios, high root porosity and density under high redox conditions. Both cushion plant species create an aerobic rhizosphere through radial oxygen loss from deep roots, which strongly reduce CH₄ fluxes to the atmosphere.

     

Exopeptidase activity in eggs of Trichostrongylus colubriformis (Nematoda)

Summary

A supernatant from eggs of the ruminant nematode Trichostrongylus colubriformis contained an enzyme that was similar to leucine aminopeptidase (LAP), based on hydrolysis of the substrate L-leucine β-naphthylamide to β-naphthylamine. A Michaelis-Menten constant (K _m) of 0.155 mM was obtained. Rate of hydrolysis of 16 substrates revealed that L-phenylalanine and L-tyrosine β-naphthylamides were hydrolyzed most readily while seven additional substrates were hydrolyzed at lesser rates. The optimum pH for enzymatic activity was 6.75–7.5. Enzymatic activity was lost by heating the egg supernatant to 60°C for 5 min or freezing at 0°C for 28 days. Addition of millimolar concentrations of the chlorides of zinc, manganese and magnesium to the egg supernatant had no stimulatory effect on enzyme activity while 10 and 100 mM concentrations significantly reduced activity. Ethylenediamine tetraacetic acid at 10^?4 M had no effect on enzymatic activity. Activity was inhibited by 10^?4 M 1,10-phenanthroline, but the inhibition was reversed by zinc chloride at 10^?3 M. Di-isopropylphosphofluoridate at 10^?3 M reduced enzymatic activity moderately. Enzyme activity in egg supernatant increased 2.2-fold from 21 days to 60–90 days of a primary infection in the host while a 3.3-fold increase was found in primary versus secondary infections.     

Optimal design for CSTR's in series using reversible Michaelis-Menten reactions

An analytical expression is derived for the optimal design of a series of CSTR's performing reversible Michaelis-Menten kinetics in the liquid phase. The optimal design is based on minimum overall volume ofN reactors in series required to achieve a certain degree of substrate conversion. The reversible Michaelis-Menten equation is also able to explain competitive product inhibition and irreversible Michaelis-Menten kinetics. The reversible Michaelis-Menten kinetics covers three types of enzymatic reactions depending on the values of the rate constant for the forward (k _s) and reverse (k _p) reactions. An optimum design is obtained in the three cases ofK _s=K_p, K_s>K_p andK _s<K_p. The minimum overall reactors volume is compared with the more convenient equal-sized CSTR's. The effect of enzyme deactivation on the minimum overall reactors volume is investigated. The performance of a series of CSTR's is compared with plug-flow reactor. Glucose isomerization which exhibits reversible Michaelis-Menten kinetics is used as a model system for optimization.     

Phytoplankton competition along a gradient of dilution rates

Natural phytoplankton from Lake Constance was used for chemostat competition experiments performed at a variety of dilution rates. In the first series at high Si:P ratios and under uniform phosphorus limitation for all species, Synedra acus outcompeted all other species at all dilution rates up to 1.6 d^-1, only at the highest dilution rate (2.0 d^-1) Achnanthes minutissima was successful. In the second series in the absence of any Si a green algal replacement series was found, with Mougeotia thylespora dominant at the lowest dilution rates, Scenedesmus acutus at the intermediate ones, and Chlorella minutissima at the highest ones. The outcome of interspecific competition was not in contradiction with the Monod kinetics of P-limited growth of the five species, but no satisfactorily precise prediction of competitive performance can be derived from the Monod kinetics because of insufficient precision in the estimate of k _s .     

Detecting Photoactivation of Phosphoenolpyruvate Carboxylase in C(4) Plants : An Effect of pH

Photoactivation of phosphoenolpyruvate carboxylase in C₄ plants is detected more efficiently when activity is assayed at suboptimum pH (e.g. 7.2); the magnitude of the light effect is often larger at low phosphoenolpyruvate concentration.

Darkness and low assay pH induce an allosteric behavior (positive cooperativity with phosphoenolpyruvate) which is relieved in light or by higher pH; thus, normal Michaelis-Menten kinetics are exhibited only when the enzyme is extracted during the day and assayed at pH 8.2.

Light activation, pH, and substrate level appear to be components of a regulatory device suppressing the activity in darkness and enhancing it under light.