Purification and properties of the tetrahydropteroylglutamate methyltransferase from green beans (Phaseolus vulgaris) (author's transl)]

The tetrahydropteroylglutamate methyltransferase from green beans (Phaseolus vulgaris) has been purified 80-fold by ion exchange chromatography and gel filtration. Optimal methyl transfer is found at pH 6.5 and 39 degrees C. Even at 0 degrees C, however, a considerable catalytic rate is observed. The Michaelis-Menten constants for homocysteine and 5-methyltetrahydropteroylglutamate are 0.43mM and 2.4 mM, respectively. Magnesium ions enhance the activity. Even purified preparations appear to contain traces of magnesium ions firmly bound, since a residual activity is found without addition of magnesium salts. Though the reaction requires anaerobiosis, an excess of reducing agents is inhibitory. The molecular weight of the transferase, determined by gel filtration, is 40 000 +/- 6%.     

Effect of low growth temperature on coupling between electron transport and proton flux in Vicia faba thylakoids

Coupling between electron transport and proton flux has been compared in chloroplasts from Vicia faba (cv. Windsor) plants grown at 20 and 5°C. Proton uptake by warm-grown thylakoids was sensitive to external pH and stimulated by micromolar adenine nucleotide above pH 7.0. Electron transport was modulated by pH, adenine nucleotide and energy transfer inhibitors (triphenyltin and Hg²⁺). By contrast, proton uptake by cold-grown thylakoids was generally lower and was insensitive to micromolar ATP. The rate of non-phosphorylating electron flow in cold-grown thylakoids was relatively insensitive to pH and Hg²⁺ and was not modulated by adenine nucleotides or triphenyltin. Stimulation of electron transport by phosphorylating conditions in cold-grown thylakoids was generally lower and insensitive to pH. It is concluded that the control of proton efflux through CF₀-CF₁ differs in thylakoids of V. faba grown at warm and cold temperatures.     

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 Km for ATP was 2.7 microM and increased little when K+ was added extravesicularly. At pH 7.5, millimolar concentrations of K+ increased the apparent Km 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.     

Acid and alkaline phosphatase activity in Trypanosoma cruzi epimastigotes

Phosphatase activity in intact Trypanosoma cruzi epimastigotes has been demonstrated. After subcellular fractionation three activities were characterized: (a) a membrane-bound microsomal acid activity with an optimum pH of 4.0 and a Km of 1.2 mM, strongly inhibited by tartrate and fluoride; (b) a soluble cytosolic acid activity with an optimum pH of 5.5 and a Km of 0.95 mM, strongly inhibited by p-hydroxymercuribenzoate, EDTA and copper ions and activated by cyanide, manganese and magnesium ions; and (c) a soluble cytosolic alkaline activity with an optimum pH of 8.0 and a Km of 3.8 mM, inhibited by p-hydroxymercuribenzoate, fluoride, EDTA, and copper, calcium and zinc ions. This activity was increased by magnesium and manganese ions.     

Cation induced changes in the rheological properties of purified mucus glycoprotein gels

The effects of mono-, di- and trivalent ions on the rheological properties of a purified mucus glycoprotein gel have been investigated. Monovalent ions increased the fluidity of the gels in a concentration dependent manner. The effect of calcium was pH dependent; at neutral pH values this ion produced a maximum in the gel viscoelasticity at a concentration of 0.5 mM, whilst at pH 5.0 the increase in viscoelasticity was sustained up to 3.0 mM. The same concentrations of copper (II) at pH 5.0 had a similar but greater effect on the viscoelasticity. Al3+ at pH 3.0 increased the viscoelastic moduli throughout the range studied (0.1-2.0 mM), whereas iron made the gels more fluid at low concentrations, but increased the viscoelasticity to above control values at a concentration of 2.0 mM.     

Isolation and characterization of an alkaline phosphatase from pea thylakoids

Endogenous dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II in pea (Pisum sativum, L. cv Progress 9) thylakoids drives the state 2 to state 1 transition; the responsible enzyme is a thylakoid-bound, fluoride-sensitive phosphatase with a pH optimum of 8.0 (Bennett J [1980] Eur J Biochem 104: 85-89). An enzyme with these characteristics was isolated from well-washed thylakoids. Its molecular mass was estimated at 51.5 kD, and this monomer was catalytically active, although the activity was labile. The active site could be labeled with orthophosphate at pH 5.0. High levels of alkaline phosphatase activity were obtained with the assay substrate, 4-methylumbelliferyl phosphate (350 micromoles per minute per milligram purified enzyme). The isolated enzyme functioned as a phosphoprotein phosphatase toward phosphorylated histone III-S and phosphorylated, photosystem II-enriched particles from pea, with typical activities in the range of 200 to 600 picomoles per minute per milligram enzyme. These activities all had a pH optimum of 8.0 and were fluoride sensitive. The enzyme required magnesium ion for maximal activity but was not dependent on this ion. Evidence supporting a putative function for this phosphatase in dephosphorylation of thylakoid proteins came from the inhibition of this process by a polyclonal antibody preparation raised against the partially purified enzyme.     

The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme-catalysed reactions.

The effects of pH and temperature on Michaelis constant (Km) and maximum velocity (Vmax.) and of NaCl on the activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase EC 3.2.1.21) from cultures of Botryodiplodia theobromae Pat. have been studied. 2. Donor binding and inhibition of activity by glucose were dependent on the ionization of a group (pK 6.0) that appeared to be an imidazole group. 3. Catalytic activity and the stimulation of activity by glycerol were dependent on the ionization of two groups, which appeared to be a carboxy group and an imidazole group. 4. The Arrhenius activation energy (Ea) calculated from results obtained at pH 4.0 and 5.0 was about 45--46kJ.mol-1. 5. The enthalpies (delta H0) calculated from results obtained at pH 4.0 and 5.0 were similar (about -4kJ.mol-1), whereas at pH 6.5 the value was about -33kJ.mol-1. 6. The entropies (delta S0) calculated from these results at 37 degrees C were -21, -22 and -118J.K-1.mol-1 at pH 4.0, 5.0 and 6.5 respectively. A low concentration of NaCl (16.6 mM) stimulated enzymic activity and decreased the Km for the donor, whereas high concentrations (up to 500 mM) inhibited enzymic activity, increased the Km and had no effect on Vmax. 8. Plots of initial velocity data obtained in the presence of dioxan as 1/v against the ratio of the molar concentration of dioxan to that of water were linear. 9. The results are discussed in terms of the enzyme mechanism.     

Modulation of serotonin uptake kinetics by ions and ion gradients in human placental brush-border membrane vesicles

The modulation of serotonin uptake kinetics by Na+, Cl-, H+, and K+ was investigated in brush-border membrane vesicles prepared from normal human term placentas. The presence of Na+ and Cl- in the external medium was mandatory for the function of the serotonin transporter. In both cases, the initial uptake rate of serotonin was a hyperbolic function of the ion concentration, indicating involvement of one Na+ and one Cl- per transport of one serotonin molecule. The apparent dissociation constant for Na+ and Cl- was 145 and 79 mM, respectively. The external Na+ increased the Vmax of the transporter and also increased the affinity of the transporter for serotonin. The external Cl- also showed similar effects on the Vmax and the Kt, but its effect on the Kt was small compared to that of Na+. The presence of an inside-acidic pH, with or without a transmembrane pH gradient, stimulated the NaCl-dependent serotonin uptake. The effect of internal [H+] on the transport function was to increase the Vmax and decrease the affinity of the transporter for serotonin. The presence of K+ inside the vesicles also greatly stimulated the initial rates of serotonin uptake, and the stimulation was greater at pH 7.5 than at pH 6.5. This stimulation was a hyperbolic function of the internal K+ concentration at both pH values, indicating involvement of one K+ per transport of one serotonin molecule. The apparent dissociation constant for K+ was 5.6 mM at pH 6.5 and 4.0 mM at pH 7.5. The effects of internal [K+] on the uptake kinetics were similar to those of internal [H+].(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromium increases photosystem 2 activity in <Emphasis Type="Italic">Brassica juncea</Emphasis>

In 7-d-old seedlings of Brassica juncea chromium (VI) promoted photosystem 2 (PS 2) mediated photoreactions. The increase in PS 2 activity in the thylakoids from Cr-treated seedlings, in the presence of uncoupler (5 mM NH₄Cl), was similar to that recorded with the control thylakoids. Thus Cr enhanced PS 2 activity was not due to uncoupling of electron transport from photophosphorylation. Photon saturation kinetics revealed that the PS 2 activity of thylakoids from Cr-treated seedlings was significantly higher at almost all irradiances in comparison to that of controls. PS 2 activity of thylakoids from Cr-treated plants at 25 % of the saturating irradiance was in par with the PS 2 activity of the thylakoids from control plants at saturating irradiance. Thylakoids from both control and Cr-treated seedlings exhibited maximum PS 2 activity at pH 7.5. The PS 2 activity of thylakoids from Cr-treated plants remained high even at pH 8.0 and 8.5, demonstrating Cr enhances tolerance of PS 2 to alkaline pH.     

Anion transport in red blood cells and arginine specific reagents. (1). Effect of chloride and sulfate ions on phenylglyoxal sensitive sites in the red blood cell membrane

Inhibition of anion transport by the arginine specific reagents phenylglyoxal and 1,2 cyclohexandione depends on the pH and anion concentration in the medium. At pH 8.0, chloride ions protect the transport system against inhibition by PG and 1,2 CHD, while sulfate ions do not protect (1). In the present paper it is shown that at pH 6.5 and 7 both sulfate ions and chloride ions protect the transport system. The protection increases with increasing concentration of the two substrate ions.     

Kinetic studies with myo-inositol monophosphatase from bovine brain

The kinetic properties of myo-inositol monophosphatase with different substrates were examined with respect to inhibition by fluoride, activation or inhibition by metal ions, pH profiles, and solvent isotope effects. F- is a competitive inhibitor versus 2'-AMP and glycerol 2-phosphate, but noncompetitive (Kis = Kii) versus DL-inositol 1-phosphate, all with Ki values of approximately 45 microM. Activation by Mg2+ follows sigmoid kinetics with Hill constants around 1.9, and random binding of substrate and metal ion. At high concentrations, Mg2+ acts as an uncompetitive inhibitor (Ki = 4.0 mM with DL-inositol 1-phosphate at pH 8.0 and 37 degrees C). Activation and inhibition constants, and consequently the optimal concentration of Mg2+, vary considerably with substrate structure and pH. Uncompetitive inhibition by Li+ and Mg2+ is mutually exclusive, suggesting a common binding site. Lithium binding decreases at low pH with a pK value of 6.4, and at high pH with a pK of 8.9, whereas magnesium inhibition depends on deprotonation with a pK of 8.3. The pH dependence of V suggests that two groups with pK values around 6.5 have to be deprotonated for catalysis. Solvent isotope effects on V and V/Km are greater than 2 and 1, respectively, regardless of the substrate, and proton inventories are linear. These results are consistent with a model where low concentrations of Mg2+ activate the enzyme by stabilizing the pentacoordinate phosphate intermediate. Li+ as well as Mg2+ at inhibiting concentrations bind to an additional site in the enzyme-substrate complex. Hydrolysis of the phosphate ester is rate limiting and facilitated by acid-base catalysis.     

Sulfide-dependent photosynthetic electron flow coupled to proton translocation in thylakoids of the cyanobacterium Oscillatoria limnetica

Light-induced proton translocation coupled to sulfide-dependent electron transport has been studied in isolated thylakoids of the cyanobacterium Oscillatoria limnetica. The thylakoids are obtained by osmotic shock of washed spheroplasts, prepared with glycine-betaine as the osmotic stabilizer. 13C NMR studies suggests that betaine is the major osmoregulator in O. limnetica. Thylakoid preparations obtained from both sulfide-induced anoxygenic cells and noninduced oxygenic cells are capable of proton pumping coupled to phenazinemethosulfate-mediated cyclic electron flow. However, only in the induced thylakoids can sulfide-dependent proton gradient (delta pH) formation be measured, using either NADP or methyl viologen as the terminal acceptor. Sulfide-dependent delta pH formation correlates with a high-affinity electron donation site (apparent Km 44 microM at pH 7.9). This site is not lost upon washing of the thylakoids. In addition, both sulfide-dependent electron transport and delta pH formation are sensitive to inhibitors of the cytochrome b6f complex such as 2-n-nonyl-4-hydroxyquinoline-N-oxide, 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol, or stigmatellin. Sulfide-dependent NADP photoreduction of low affinity (which does not saturate by as much as 7 mM sulfide) is detected in both induced and noninduced thylakoids, but this activity is insensitive to the inhibitors and is not coupled to proton transport. It is suggested that the adaptation of O. limnetica to anoxygenic photosynthesis involves the induction of a thylakoid factor(s) which creates a high-affinity site for sulfide, and the transfer of its electrons via the cytochrome b6f complex, coupled to proton translocation.     

Improvement of biohydrogen production by Enterobacter cloacae IIT-BT 08 under regulated pH

The present study investigates the effect of pH and intermediate products formation on biological hydrogen production using Enterobacter cloacae IIT-BT 08. Initial pH was found to have a profound effect on hydrogen production potential, while regulating the pH 6.5 throughout the fermentation was found to increase the cumulative hydrogen production rate and yield significantly. Modified Gompertz equation was used to fit the cumulative hydrogen production curves to obtain the hydrogen production potential P, the hydrogen production rate R and lag phase λ. At regulated pH 6.5, higher H(2) yield (3.1molH(2)mol(-1) glucose), specific hydrogen production potential (798.1mL/g) and specific rate of H(2) production (72.1mLL(-1)h(-1)g(-1)) were obtained. The volatile fatty acid profile showed butyrate, ethanol and acetate as the major end metabolites of fermentation under the operating pH conditions tested; however, their pattern of distribution was pH dependent. At the optimum pH of 6.5, the acetate to butyrate ratio (A/B ratio) was found to be higher than that at any other pH. The study also investigates the effect of sodium ions on biohydrogen production potential. It was also found that sodium ion concentration up to 250mM enhanced the hydrogen production potential; however, any further increase in the metal ion concentration had an inhibitory effect.     

Acidophilic haloarchaeal strains are isolated from various solar salts

Haloarchaeal strains require high concentrations of NaCl for their growth, with optimum concentrations of 10–30%. They display a wide variety of morphology and physiology including pH range for growth. Many strains grow at neutral to slightly alkaline pH, and some only at alkaline pH. However, no strain has been reported to grow only in acidic pH conditions within the family Halobacteriaceae. In this study, we isolated many halophiles capable of growth in a 20% NaCl medium adjusted to pH 4.5 from 28 commercially available salts. They showed growth at pH 4.0 to 6.5, depending slightly on the magnesium content. The most acidophilic strain MH1-52-1 isolated from an imported solar salt (pH of saturated solution was 9.0) was non-pigmented and extremely halophilic. It was only capable of growing at pH 4.2–4.8 with an optimum at pH 4.4 in a medium with 0.1% magnesium chloride, and at pH 4.0–6.0 (optimum at pH 4.0) in a medium with 5.0% magnesium. The 16S rRNA and DNA-dependent RNA polymerase subunit B' gene sequences demonstrated clearly that the strain MH1-52-1 represents a new genus in the family Halobacteriaceae.     

P680(+)* reduction kinetics and redox transition probability of the water oxidizing complex as a function of pH and H/D isotope exchange in spinach thylakoids.

The rise of fluorescence as an indicator for P680(+)* reduction by YZ and the period-four oscillation of oxygen yield induced by a train of saturating flashes were measured in dark-adapted thylakoids as a function of pH in the absence of exogenous electron acceptors. The results reveal that: (i) the average amplitude of the nanosecond kinetics and the average of the maximum fluorescence attained at 100 micros after the flash in the acidic range decrease with decreasing pH; (ii) the oxygen yield exhibits a pronounced period-four oscillation at pH 6.5 and higher damping at both pH 5.0 and pH 8.0; (iii) the probability of misses in the Si-state transitions of the water oxidizing complex is affected characteristically when exchangeable protons are replaced by deuterons [at pH <6.5, the ratio alpha(D)/alpha(H) is larger than 1 whereas at pH >7.0 values of <1 are observed]. The results are discussed within the framework of a combined mechanism for P680(+)* reduction where the nanosecond kinetics reflect an electron transfer coupled with a "rocket-type" proton shift within a hydrogen bridge from YZ to a nearby basic group, X [Eckert, H.-J., and Renger, G. (1988) FEBS Lett. 236, 425-431], and subsequent relaxations within a network of hydrogen bonds. It is concluded that in the acidic region the hydrogen bond between YZ and X (most likely His 190 of polypeptide D1) is interrupted either by direct protonation of X or by conformational changes due to acid-induced Ca2+ release. This gives rise to a decreased P680(+)* reduction by nanosecond kinetics and an increase of dissipative P680(+)* recombination at low pH. A different mechanism is responsible for the almost invariant amplitude of nanosecond kinetics and increase of alpha in the alkaline region.     

Electrostatic Interaction between Plastocyanin and P700 in the Electron Transfer Reaction of Photosystem I-Enriched Particles

The nature of the electron transfer reaction between reducedplastocyanin and P700 oxidized by flash illumination was studiedin P700-enriched Triton subchloroplast fraction 1 particles.An addition of monovalent salts to the suspension at neutralpH increased the reaction rate at low concentrations (>20mM). Salts of divalent cations showed a similar effect at muchlower concentrations (>2 mM), This effect was not dependenton the concentration and the valence of anions. The increaseof rate at low salt concentrations was observed at pH's above5, but below pH 5 the rate was decreased by adding salts. Atabout pH 5, the rate was not affected by salts. Apart from thesesalt effects, the optimum pH for the reaction rate was observedbetween 5.5 and 6.5. The reduction rate depended sigmoidally on the added plastocyaninconcentration at pH 6.8 and 4. A Michaelis-Menten type relationshipwas observed at about pH 5. The half-saturation concentrationof plastocyanin became lower as the salt concentration increasedat pH 6.8, while it became higher by adding salt at pH 4. The effects of salts on the rate of electron donation from othermetalloproteins and artificial electron donors to P700 werealso studied. It is concluded, from the analysis with the Gouy-Chapmantheory, that the net charges on the electron donors and themembrane surfaces mainly determine the response of the P700reduction rate to salt addition. The salt addition changes mainlythe local concentration or accessibility of electron donorsto P700. (Received January 12, 1981; Accepted March 6, 1981)     

Effects of heterocyclic and tertiary permeant amines on the electron transfer in thylakoid membranes

The effect of low concentrations (up to 50 μM) of lipophilic permeant amines on the electron transfer in thylakoid membranes of pea chloroplasts has been investigated. In the presence of heterocyclic amines (9-aminoacridine and neutral red), the electron transfer, initiated from H₂O to PS I acceptors, has been shown to be inhibited to a level amounting to less than 50% of control, this taking place for both the basal (at alkaline pH) and the gramicidin-uncoupled transport (at pH 6.5–8.5). Under the same conditions, tertiary amines (dibucaine, tetracaine) cause only a 10–15% inhibition of transport. With all the amines, the rate of electron transport from H₂O to DCBQ, PS II acceptor is decreased to 80–90% of control at pH above 8.0, but this effect is completely removed when pH is lowered to 7.7–6.5. In the region of PS I, all the amines accelerate the basal transport, but do not influence the uncoupled electron transfer. A conclusion has been drawn that, parallel with uncoupling, heterocyclic and tertiary amines also cause an inhibition of PS II, appearing at alkaline pH values. Additionally, heterocyclic amines seem to brake electron flow at the level of plastoquinone reduction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ferredoxin-Dependent Photosynthetic Electron Transport and Coupled Processes of Energy Storage in Chloroplasts of Wheat Plants Grown under Different Levels of Nitrogen Supply

The rates of electron transfer in the presence of natural cofactors, ferredoxin and NADP, which were added in the amounts catalyzing noncyclic or cyclic electron transfer, were studied in thylakoids isolated from 17-day-old wheat seedlings. Upon excitation of both photosystems (PS) of photosynthesis, the potential rate of NADP reduction in thylakoids isolated from plants grown on nitrogen-free nutrient solution did not differ from that in thylakoids from the control plants. However, the P/2e ratio was significantly lower in thylakoids isolated from nitrogen-deficient plants. On the contrary, in the presence of DCMU, the rate of PSI-driven electron transfer from an artificial donor to NADP was considerably higher in these than in the control thylakoids. In the presence of ferredoxin under anaerobic conditions, the rate of phosphorylation coupled to cyclic electron transport was also significantly higher in thylakoids isolated from nitrogen-deficient plants, than in thylakoids isolated from control plants. Our data show that PSI-driven electron transport and cyclic photophosphorylation are activated in nitrogen-starved wheat plants, at least at the initial stages of starvation.     

Electrochemical consideration on the optimum pH of bilirubin oxidase

Steady-state current-potential curves were obtained for the direct electron transfer (DET) of bilirubin oxidase (BOD) at a highly oriented pyrolytic graphite electrode, and the theoretical analysis based on nonlinear regression enabled us to determine the formal redox potential (E degrees') of BOD in a wide pH range of 2.0 to 8.5. Cyclic voltammetric measurements were also performed for substrates, including p-phenylenediamine (PPD), o-aminophenol (OAP), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and their E degrees ' values or the anodic peak potentials (for OAP) were determined at various pH values. The difference in the redox potentials between BOD and substrates (DeltaE degrees') showed a maximum at pH 6.5 to 8.0, pH 6.5 to 8.0, and pH 3.5 to 4.5 for PPD, OAP, and ABTS, respectively. These pH ranges should be thermodynamically most favorable for the electron transfer between BOD and the respective substrates. In practice, the pH ranges showing a maximum DeltaE degrees' corresponded well with the optimum pH values for the O(2) reduction activity of BOD: pH 6.5 to 7.5, pH 8.0 to 8.5, and pH 4.0 for PPD, OAP, and ABTS, respectively. Thus, it was suggested that DeltaE degrees ' should be one of the primary factors determining the activity of BOD with the substrates.