Kinetic properties of glutaminase from cerebral cortex

The rates of phosphate activated glutaminase activity in finely homogenised cerebral cortex and synaptosomes were measured. Activity was 25–50% higher at pH 7.0 than at pH 8.0. Glutamate inhibited activity with a K_i of 2–3 mM while aspartate had little effect. Calcium (1 mM) activated the enzyme but magnesium was without action. The pH profiles of the effects of these modulators of glutaminase activity in these finely ground preparations showed that all agents were more effective at pH 7.0 than at pH 8.0.Dedicated to Henry McIlwain.     

Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruit

In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K _m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose (K _m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato fruit glucokinase was sensitive to product inhibition by ADP (K _i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate. Received: 4 October 1997 / Accepted: 10 January 1998     

1-Hydroxycyclopropane carboxylic acid phosphate: A potent inhibitor of enzymes metabolizing phosphoenolpyruvate

1-Hydroxycyclopropane carboxylic acid phosphate has been synthesized from diethyl succinate by acyloin condensation followed by ring contraction and phosphorylation. This compound is a potent competitive inhibitor of enzymes utilizing phosphoenolpyruvate. For phosphoenolpyruvate from maize, K_i = 7.3 μM at pH 8.0 in the presence of Mg²⁺. For pyruvate kinase, K_i = 2.0 mM at pH 7.0. For enolase, K_i = 8.0 μM at pH 8.0. In each case, this compound is a substantially better inhibitor than the commonly used phosphoenolpyruvate analogs phosphoglycolate and phospholactate, presumably because of the similarity in geometric and electronic structure between the cyclopropane compound and phosphoenolpyruvate.     

Hydration of 3-cyanopyridine to nicotinamide by crude extract nitrile hydratase

Summary The kinetic and stability characteristics of crude extract nitrile hydratase fromBrevibacterium R-312 were studied for the hydration of 3-cyanopyridine to nicotinamide. The enzyme was substrate and product inhibited and had the following kinetic constants:K _m =28 mM;K _p =36 mM;K _s =155 mM;V _m =5.8 mol/min/mg protein (25°C). Itsmaximum temperature and pH (phosphate buffer) were 35°C and 8.0, respectively and it had half-lives of 50 days, 10 days and 1 day at 4°C, 10°C and 25°C, respectively. The crude extract also exhibited amidase activity on nicotinamide, but it became significant only at nicotinamide concentrations greater than 300 mM. Mathematical models for batch and fed-batch hydrations were developed to account for substrate and product inhibitions and for enzyme decay. They predicted to within 10% experimental results for initial substrate and final product concentrations up to 300 mM; the accuracies decreased at higher concentrations primarily because of the relatively rapid hydrolysis of nicotinamide.     

Production and characterization of thermostable alkaline phytase from <Emphasis Type="Italic">Bacillus laevolacticus</Emphasis> isolated from rhizosphere soil

A novel phytase producing thermophilic strain of Bacillus laevolacticus insensitive to inorganic phosphate was isolated from the rhizosphere soil of leguminous plant methi (Medicago falacata). The culture conditions for production of phytase by B. laevolacticus under shake flask culture were optimized to obtain high levels of phytase (2.957 ± 0.002 U/ml). The partially purified phytase from B. laevolacticus strain was optimally active at 70 °C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with ∼80% activity at 70 °C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl₂. The phytase from B. laevolacticus showed high specificity for phytate salts of Ca⁺ > Na⁺. The enzyme showed an apparent K _m 0.526 mM and V _max 12.3 μmole/min/mg of activity against sodium phytate.     

Regulatory characteristics of a fructose bisphosphatase from the blue-green bacterium Anacystis nidulans.

A specific fructose 1,6-bisphosphatase (EC 3.1.3.11) has been partially purified from the obligately autotrophic blue-green bacterium Anacystis nidulans. It was most active at pH 8.0. The K_m for fructose 1,6-bisphosphate was 0.088 mm at pH 8.0 and 0.105 mm at pH 7.0; the K_m for MgCl₂ was 0.95 mm at pH 8.0. Activity at netural pH was particularly sensitive to the MgCl₂ concentration. AMP was an allosteric inhibitor, 50% inhibition being exerted by 0.058 mm AMP at pH 7.0 and 0.085 mm AMP at pH 8.O. The way in which changes in intracellular pH and the concentrations of Mg²⁺ and AMP might influence the activity of the enzyme in the Calvin cycle, the oxidative pentose phosphate pathway and in glycolysis and gluconeogenesis is discussed.     

A novel xylose isomerase from Neurospora crassa

Summary Highest production of xylose Isomerase by Neurospora crassa grown with different carbon sources was at 0.014 U mg^-1 with D-xylose. The enzyme exhibited maximum activity at pH 8.0 and 70°C and retained 100% activity at 45°C for 30 min at pH 8.0. It was activated by 8 mM Mg²⁺ whereas 2 mM Co²⁺ afforded protection against inactivation by heat. The K _m for xylose was 10 mM and 22 mM for xylose Isomerase and xylose reductase respectively at 28°C and pH 7.0. This is the first report on the presence of xylose isomerase in N. crassa and the existence of two different pathways for the utilization of D-xylose.     

Purification and Properties of Gitrus natsudaidai Pectinesterase

The level of glutamine synthetase in Micrococcus glutamicus ATCC 13032 varied in response to the nitrogen source in culture medium; it was 10?20 fold higher in glutamate-, peptone- or yeast extract-grown cells than in ammonia- or urea-grown cells. Ammonia (3 mM) reduced the enzyme level to 50% when added to glutamate medium. No difference between nitrogen sources was observed in extent of inhibition by Mg²⁺ of γ-glutamylhydroxamate-forming (transferring) reaction in crude extracts.

The optimum pH was 7.0 ? 8.0 for glutamine-forming (synthesizing) reaction and 7.0 for transferring reaction. The enzyme was stable to heating at 50°C for 10 min in 0.05 M potassium phosphate buffer (pH 6.0) containing 0.1 mM MnCl₂. Km values for glutamate, ammonia and ATP in synthesizing reaction were 7.9, 5.0 and 1.2 mM, respectively. GTP and hydroxylamine could be substituted for ATP and ammonia with about 10 and 30% reactivity. Mg²⁺ was effective as a cofactor in synthesizing reaction and Mn²⁺ showed 34% of the reactivity of Mg²⁺ at a concentration of 30 mM. Glutamine synthetase was inhibited by adenosine, AMP and ADP but not by amino acids other than D-threonine. The regulation system of glutamine synthetase in M. glutamicus is discussed.     

Factors Affecting Glucose and Maltose Phosphorylation by the Ruminal Bacterium Megasphaera elsdenii

The objectives of this study were to examine the effects of growth substrate and extracellular pH on phosphoenolpyruvate-dependent glucose phosphorylation as well as to examine how maltose is phosphorylated by the ruminal bacterium Megasphaera elsdenii B159. Phosphoenolpyruvate-dependent glucose phosphorylation by toluene-treated cells was constitutive, and glucose phosphorylation was reduced by 69% at pH 5.0. When toluene-treated cells were incubated in histidine buffer, little maltose phosphorylation occurred in the absence of inorganic phosphate. However, the addition of increasing concentrations of either potassium or sodium phosphate increased maltose phosphorylation. Maximal phosphorylation activity was observed at between 25 and 50 mM of either inorganic phosphate source. Compared with the control incubations, maltose phosphorylation was increased over threefold with 25 mM of either potassium or sodium phosphate. Phosphoglucomutase activity was detected in cell extracts of M. elsdenii B159, and this enzyme had a K _m of 3.2 mM for glucose-1-P and a V _max of 1836 nmol of NADP⁺ reduced/mg of protein per min. Maltose was also hydrolyzed by an inducible maltase (K _m , 1.19 mM). To our knowledge, this is the first report of a maltose phosphorylase and a maltase in M. elsdenii. Received: 3 November 1999 / Accepted: 5 January 2000     

Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.     

Species specific release of sulfate from adenylyl sulfate by ATP sulfurylase or ADP sulfurylase in the green sulfur bacteria Chlorobium limicola and Chlorobium vibrioforme

High activities of ATP sulfurylase were found in the soluble protein fraction of two Chlorobium limicola strains, whereas ADP sulfurylase was absent. ATP sulfurylase was partially purified and characterized. It was a stable soluble enzyme with a molecular weight of 230,000, buffer-dependent pH optima at 8.6 and 7.2 and an isoelectric point at pH 4.8. No physiological inhibitor was found. Inhibition was observed with p-CMB and heavy metals. Sulfur compounds had no effect on enzyme activity. The stoichiometry of the reaction was proven. In contrast, an ADP sulfurylase, but no ATP sulfurylase, was found in Chlorobium vibrioforme. This enzyme was very labile with a molecular weight of about 120,000 and buffer-dependent pH optima at 9.0 and 8.5. Under test conditions the apparent K _m value was determined to be 0.28 mM for adenylyl sulfate and 8.0 mM for phosphate.Abbreviations APS adenylyl sulfate - p-CMB parachloromercuribenzoate - PP_i inorganic pyrophosphate     

K+ channels of stomatal guard cells: bimodal control of the K+ inward-rectifier evoked by auxin

The influence of the auxins indole-3-acetic acid (IAA) and 1-napthylene acetic acid (NAA) on K⁺ channels and their control was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes to record membrane potentials and to monitor K⁺ channel currents under voltage clamp during exposures to 0.1–100 µM IAA and NAA. Following impalements, challenge with either IAA or NAA in the presence of 10 mM KCl resulted in the concerted modulation of at least four different currents with distinct kinetic characteristics and concentration dependencies. Equivalent concentrations of benzoic acid were wholly without effect. Most striking, current carried by inward-rectifying K⁺ channels (I_K,in) exhibited a bimodal response to both IAA and NAA which was reversed on washing the auxins from the bathing medium. The steady-state current was augmented 1.3- to 2-fold at concentrations between 0.1 and 10 µM and antagonized at concentrations near 30 µM and above. Auxin agonism of I_K,in was time- and voltage-independent. By contrast, I_K,in inactivation at the higher auxin concentrations was marked by a voltage-dependence and slowing of the kinetics for current activation. Inactivation of I_K,in by the auxins was relieved when cytoplasmic pH (pH_i) was clamped near 7.0 in the presence of 30 mM Na⁺-butyrate. In addition to the control of I_K,in, current carried by a second class of (outward-rectifying) K⁺ channels rose in a monotonic and largely voltage-independent manner with auxin concentrations about 10 µM and above, and IAA and NAA also activated an inward-going current with a voltage dependence characteristic of guard cell anion channels. Further changes in background current were consistent with a limited activation of the H⁺-ATPase. Over the concentration range examined, the auxins evoked membrane hyperpolarizations and depolarizations of up to ±12–19 mV, depending on the free-running membrane potential prevailing before auxin additions. Prolonging exposures to 100 µM auxin beyond 3–5 min frequently elicited rapid transitions to voltages near E_K as well as regenerative action potentials. However, in every case the voltage response was a predictable consequence of auxin action on the K⁺ channels and, at 100 µM auxin, on the anion current. These results demonstrate a control of K⁺ channel activity by auxin, consistent with the roles of these channels in mediating K⁺ flux for stomatal movements; the data associate a bimodal characteristic with the activity of I_K,in, implicating pH_i as a putative intermediate in its control, and offer strong evidence for a multiplicity of signal cascades evoked by auxin; finally, they highlight a coordinate modulation of transport activities by auxin, thereby drawing a close analogy to the pattern of stimulus-response coupling in abscisic acid.     

Membrane-bound nitrite oxidoreductase of Nitrobacter: evidence for a nitrate reductase system

Nitrite oxidoreductase, the essential enzyme complex of nitrite oxidizing membranes, was isolated from cells of the nitrifying bacterium Nitrobacter hamburgensis. The enzyme system was solubilized and purified in the presence of 0.25% sodium deoxycholate. Nitrite oxidoreductase oxidized nitrite to nitrate in the presence of ferricyanide. The pH optimum was 8.0, and the apparent K _m value for nitrite amounted to 3.6 mM. With reduced methyl-and benzylviologen nitrite oxidoreductase exhibited nitrate reductase activity with an apparent K _m value of 0.9 mM for nitrate. NADH was also a suitable electron donor for nitrate reduction. The pH optimum was 7.0.Treatment with SDS resulted in the dissociation into 3 subunits of 116,000, 65,000 and 32,000. The enzyme complex contained iron, molydbenum, sulfur and copper. A c-type cytochrome was present. Isolated nitrite oxidoreductase is a particle of 95±30 Å in diameter.Abbreviation DOC sodium deoxycholate     

Separation and characterization of two chorismate-mutase isoenzymes from Nicotiana silvestris

Two isoenzymes of chorismate mutase (EC 5.4.99.5) were isolated and partially purified from leaves of diploid (2n=24) Nicotiana silvestris Speg. et Comes and from isogenic cells in a suspension culture originally established from haploid tissue. An isoenzyme denoted CM-1 (M _r=52,000) accounted for the major fraction of total activity recovered from suspension-cultured cells, while isoenzyme CM-2 (M _r=65,000) represented the major fraction of activity recovered from green leaf tissue. The ratio of isoenzyme levels from these two sources differed more than 20-fold. The subcellular location of isoenzyme CM-1 is known to be in the chloroplasts of green leaves or in proplastids of cultured cells, while isoenzyme CM-2 is located in the cytosol. Both isoenzymes were stable during partial purification, possessed broad pH optima for catalysis between 6.0 and 8.0, and were active without denaturation at temperatures at least as high as 45° C. Thiol reagents were unnecessary for either stability or activity of both isoenzymes. The affinity of isoenzyme CM-2 for substrate (K _m=0.24 mM) was almost an order of magnitude better than that of CM-1. The kinetic behavior of isoenzyme CM-1 was influenced by pH, while that of isoenzyme CM-2 was not. At pH 7.2, hyperbolic substrate-saturation curves (K _m=1.7 mM) were obtained for isoenzyme CM-1. At pH 6.1, however, isoenzyme CM-1 displayed relatively weak positive cooperativity, Hill plots yielding an n value of 1.2 At pH 6.1 the half-saturation ([S]_0.5) value was 2.5 mM.Abbreviations DEAE diethylaminoethyl - M _r molecular weight     

Novel NADPH-dependent L-aspartate dehydrogenases from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum

The genes encoding putative L-aspartate dehydrogenases (EC 1.4.1.21, ADH) from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum were cloned and expressed in Escherichia coli. The respective enzymes in the form of hybrid proteins with N-terminal hexahistidine tags were purified to apparent homogeneity. Both enzymes catalyzed in vitro the reductive amination of oxaloacetate to L-aspartate by an order faster than the reverse reaction at a respective pH optimum of 8.0–9.0 and 9.8; also, the enzymes only catalyzed amination under physiological conditions (pH 7.0–8.0). Their specificity to NADPH was higher by 1–2 orders of magnitude than that to NADH. The apparent K _M values of ADH from R. palustris for oxaloacetate, ammonium, and NADPH at pH 9.0 were 9.2, 11.3, and 0.21 mM, respectively, and the corresponding K _M values of ADH from B. japonicum were 21, 4.3, and 0.032 mM, respectively. The amination activity of novel ADHs may be important for the fixation of inorganic nitrogen in vivo and used for the construction of a bacterial strain-producer of L-aspartate by metabolic engineering methods.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Effect of the manganese ion on human α3/4 fucosyltransferase III activity

The effect of manganese and other divalent cations on the activity of a soluble recombinant form of human 3/4 fucosyltransferase III (SFT3) expressed in Spodoptera frugiperda (Sf9) insect cells was studied. SFT3 was active in the absence of divalent cations with an optimum pH of 4.5. In the absence of Mn²⁺ increasing the pH from 4.5 to 7.0 caused a decrease in the affinity of SFT3 for the acceptor Gal3GlcNAcO(CH₂)₃NHCO(CH₂)₅NH-biotin, as monitored by the 4-fold increase in the apparent K_M value (0.9 to 3.3 mM). At pH 7.0, the addition of Mn²⁺ activated the enzyme and caused an increase in the affinity of SFT3 for the acceptor, as monitored by the 5-fold decrease of the apparent K_M value (3.3 to 0.7 mM). In solution, a complex between GDP-Fuc donor and the divalent cation Mn²⁺ was observed by electrospray ionization mass spectrometry, in a 1:1 stoichiometry. These results indicated that Mn²⁺ bound the enzyme and increased its affinity for the acceptor; one possible functional role of manganese in catalysis could be as an electrophilic catalyst, co-ordinating the negative charges of the phosphate groups of the GDP-Fuc donor and promoting Fuc transfer. At low pH values such role would be played by the proton.     

An equilibrium binding study of the interaction of fructose 6-phosphate and fructose 1,6-bisphosphate with rabbit muscle phosphofructokinase.

Equilibrium binding studies of the interaction of rabbit muscle phosphofructokinase with fructose 6-phosphate and fructose 1,6-bisphosphate have been carried out at 5 degrees in the presence of 1-10 mM potassium phosphate (pH 7.0 and 8.0), 5 mM citrate (pH 7.0), or 0.22 mm adenylyl imidodiphosphate (pH 7.0 and 8.0). The binding isotherms for both fructose 6-phosphate and fructose 1,6-bisphosphate exhibit negative cooperativity at pH 7.0 and 8.0 in the presence of 1-10 mM potassium phosphate at protein concentrations where the enzyme exists as a mixture of dimers and tetramers (pH 7.0) or as tetramers (pH 8.0) and at pH 7.0 in the presence of 5 mM citrate where the enzyme exists primarily as dimers. The enzyme binds 1 mol of either fructose phosphate/mol of enzyme monomer (molecular weight 80,000). When enzyme aggregation states smaller than the tetramer are present, the saturation of the enzyme with either ligand is paralleled by polymerization of the enzyme to tetramer, by an increase in enzymatic activity and by a quenching of the protein fluorescence. At protein concentrations where aggregates higher than the tetramer predominate, the fructose 1,6-bisphosphate binding isotherms are hyperbolic. These results can be quantitatively analyzed in terms of a model in which the dimer is associated with extreme negative cooperativity in binding the ligands, the tetramer is associated with less negative cooperativity, and aggregates larger than the tetramer are associated with little or no cooperativity in the binding process. Phosphate is a competitive inhibitor of the fructose phosphate sites at both pH 7.0 and 8.0, while citrate inhibits binding in a complex, noncompetitive manner. In the presence of the ATP analog adenylyl imidodiphosphate, the enzyme-fructose 6-phosphate binding isotherm is sigmoidal at pH 7.0, but hyperbolic at pH 8.0. The characteristic sigmoidal initial velocity-fructose 6-phosphate isotherms for phosphofructokinase at pH 7.0, therefore, are due to an heterotropic interaction between ATP and fructose 6-phosphate binding sites which alters the homotropic interactions between fructose 6-phosphate binding sites. Thus the homotropic interactions between fructose 6-phosphate binding sites can give rise to positive, negative, or no cooperativity depending upon the pH, the aggregation state of the protein, and the metabolic effectors present. The available data suggest the regulation of phosphofructokinase involves a complex interplay between protein polymerization and homotropic and heterotropic interactions between ligand binding sites.     

Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK _m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD⁺. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK _m value of 1.2 mM and a high requirement of Mg²⁺ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK _m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD⁺. For the non-specific HK, only theK _m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase     

Replacement of potassium ions by ammonium ions in different micro-organisms grown in potassium-limited chemostat culture

The biomass concentration extant in potassiumlimited cultures of either Klebsiella pneumoniae or Bacillus stearothermophilus (when growing at a fixed temperature and dilution rate in a glucose/ammonium salts medium) increased progressively as the medium pH value was raised step-wise from 7.0 to 8.5. Because the macromolecular composition of the organisms did not vary significantly, this increase in biomass could not be attributed to an accumulation of storage-type polymers but appeared to reflect a pH-dependent decrease in the cells' minimum K⁺ requirement. Significantly, this effect of pH was not eviden with cultures in which no ammonium salts were present and in which either glutamate or nitrate was added as the sole nitrogen source; however, it was again manifest when various concentrations of NH₄Cl were added to the glutamate-containing medium. This suggested a functional replacement of K⁺ by NH ₄ ⁺ , a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 Å) and the ammonium ion (1.43 Å). At pH 8.0, and with a medium containing both glutamate (30 mM) and NH₄Cl (100 mM), cultures of B. stearothermophilus would grow without added potassium at a maximum rate of 0.7 h^-1. Under these conditions the cells contained maximally 0.1% (w/w) potassium (derived from contaminating amounts of this element in the medium constituents), a value which should be compared with one of 1.4% (w/w) for cells growing in a potassiumlimited medium containing initially 0.5 mM K⁺. Qualitatively similar findings were made with cultures of K. pneumoniae; and whereas one may not conclude that NH ₄ ⁺ can totally replace K⁺ in the growth of these bacteria, it can clearly do so very extensively.