The effects of high concentrations of salts on photosynthetic electron transport of immobilized thylakoids: Functional stability

Summary Stability studies of photosynthetic activity under continuous saturating illumination are presented. Chloroplast membranes (thylakoids) are isolated in a classical Hepes/sorbitol buffer or in high salt concentration buffers (citrate or sulphate) and then immobilized in a co-crosslinking serum albumin-glutaraldehyde matrix. The activities of these immobilized systems tested in a batch reactor are greatly increased by high concentrations of salts (223 and 277 mol ferrocyanide/mg of chlorophyll per hour for citrate; 243 and 267 mol ferrocyanide/mg of chlorophyll per hour for sulphate, compared with 141 mol ferrocyanide/mg of chlorophyll per hour for sorbitol). In continuous stirred-tank reactors, the conversion rates increase when high concentrations of salts are present in the buffer (approximately 36% for citrate and 34% for sulphate compared with 18% for sorbitol). The functional stability of these immobilized systems during continuous illuminations is higher in citrate (7.5 h) than in sulphate (5.5 h) or sorbitol (3.5 h). These experiments performed in batch or in continuous stirred-tank reactors underline the importance of salt ions in the reaction media.Abbreviations ADP Adénosine diphosphate - ATP Adénosine triphosphate - EDTA Ethylenediaminetetraacetate - Hepes 4-(2-hydroxyethyl)-1 piperazine-ethane sulphonic acid - Sorbitol thylakoids thylakoids isolated in sorbitol buffer - Citrate thylakoids thylakoids isolated in potassium citrate buffer - Sulphate thylakoids thylakoids isolated in sodium sulphate buffer - Immobilized sorbitol thylakoids thylakoids isolated in sorbital buffer and then immobilized in an albumin matrix - Immobilized citrate thylakoids thylakoids isolated in potassium citrate buffer and then immobilized in an albumin matrix - Immobilized sulphate thylakoids thylakoids isolated in sodium sulphate buffer and then immobilized in an albumin matrix - Control thylakoids thylakoids isolated in sorbitol buffer and tested in sorbitol buffer - High salt thylakoids thylakoids isolated in high salt concentration buffer and tested in this buffer     

Modulation by a high citrate concentration of kinetic parameters and of functional stability of two immobilized thylakoid systems

This article deals with aspects of the reciprocal interaction between the activity of chloroplast membranes and their microenvironment. The artificial matrices used in the present work to immobilize thylakoids (albumin-glutaraldehyde matrix, polyurethane foam) can be regarded as weak ion exchangers. Thus, the distribution of the solute between the matrix surface and the external solution should, at least in part, be governed by a Donnan equilibrium. The influence of a high ionic strength medium (750mM potassium citrate) on the kinetic parameters (K(p1) V(m)) and on the stability of the photosynthetic activity of immobilized chloroplast membranes has been studied. The results show similarities in behavior of the two supports studied in that, for both, a high concentration of salt (citrate) increases the apparent affinity for ferricyanide and allows a better transformation of this electron acceptor in CSTR experiments.     

Influence of immobilization procedure and salt environment on functional stability of chloroplast membranes: experimental data and numerical analysis

The interactions between chloroplast membranes and their microenvironment within artificial matrices (albumin-glutaraldehyde matrix, polyurethane foam) where investigated. Particularly, the influence of a high-ionic-strength medium (0.75 M potassium citrate) on the stability of the photosynthetic ferricyanide reduction by immobilized thylakoids has been studied. A method of data analysis based on a nonlinear identification method combined with the numerical integration of the equation of the transient state of the continuous stirred tank reactor (CSTR) is proposed to estimate the actual degradation of the photosynthetic electron transfer. A statistical analysis achieved on the parameter values has allowed a quantitative assessment of the global behavior of immobilized chloroplast membranes. From the mathernatical analysis of the experimental data, we demonstrate that citrate used in the reaction media prevents the photoinactivation of the electron transfer chain whatever the nature of the matrix or the type of the reactor. The use of an albumin-glutaraldehyde matrix or an open reactor during experiments also has allowed a better stabilization of the photosystems under operational conditions.     

The effects of oxygen solubility and high concentrations of salts on photosynthetic electron transport in chloroplast membranes.

Chloroplast membranes were isolated in different media containing Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid] and high concentrations of sorbitol (0.33 M), potassium citrate (0.75 M) or Na2SO4 (1.0 M). Due to the complexity of the media, the oxygen solubility is strongly modified by high concentrations of salts (oxygen solubility for 0.33 M-sorbitol, 0.21 mmol/litre; for 0.75 M-potassium citrate, 0.121 mmol/litre; and for 1.0 M-Na2SO4, 0.112 mmol/litre). The knowledge of these values is necessary to interpret the rate of O2 evolution. For thylakoids isolated in 'sorbitol buffer' and then tested in high concentrations of potassium citrate, a slight stimulation of O2 evolution is observed (143-173 mumol of O2/h per mg of chlorophyll a) with potassium ferricyanide as electron acceptor. When we monitor the potassium ferricyanide reduction, no stimulation of electron transport is obtained even if the observed phenomenon is identical with the Photosystem-II oxygen evolution. In the same experiments no stimulation of the photophosphorylation was recorded, but when thylakoids are directly isolated in 0.75 M-potassium citrate, O2 evolution, ferricyanide reduction and photophosphorylation are inhibited by high concentrations of salts. The behaviour of thylakoids seems to be influenced by their initial treatment.     

Effect of the filamentous structure of myosin on the actomyosin ATPase activity

The ATPase activities of acto-heavy meromyosin and of acto-myosin minifilaments have been compared under the same conditions at low ATP (0.1 mM) and at several KC1 concentrations. The activities, which are strongly salt-dependent in both systems, have been found to be similar at high ionic strength (about 0.16 M) but different at lower ionic strength (0.06-0.07 M). Under this last condition, the catalytic constants kcat and Km are lower for acto-myosin minifilaments than for acto-heavy meromyosin ATPase. In addition, at low ionic strength, any decrease in the concentration of any of the ionic species (ATP, citrate, etc.) induces an increase in the interaction strength between myosin and actin filaments, as revealed by the Km changes. The presence of the troponintropomyosin complex and of Ca2+ also enhances the strength of this interaction. On the other hand, the occurrence of particular interactions between F-actin and myosin minifilaments is further substantiated by the phenomenon of superprecipitation which occurs when the ATP concentration decreases. The favourable effect of the organized structure of the myosin minifilaments on the ATPase activity of actomyosin is discussed.     

The sulphatase of ox liver. XIX. On the nature of the polymeric forms of sulphatase A present in dilute solutions.

Weight-average elution volumes of sulphatase A (an arylsulphate sulphohydrolase, EC 3.1.6.1) from Sephadex G-200 have been determined as functions of protein concentration, pH, ionic strength and temperature. The results are used to calculate the apparent association equilibrium constants for tetramer formation and the associated standard-state thermodynamic parameters. While the apparent association constant decreased from 10(28) to 10(21) M-3 on increasing the pH from 4.5 to 5.6 at ionic strength 0.1, at any particular pH value studied it was relatively insensitive to temperature variation so that deltaH is close to zero and tetramer formation in solution is associated with a positive entropy change. At pH 5.0, increasing the ionic strength from 0.1 to 2 decreased the association constant by a factor of 100. Methylumbelliferone sulphate has no effect on the association of sulphatase A. The equilibrium results are used to define the degree of association of sulphatase A likely to encountered in experiments designed to elucidate its kinetic properties. In the liver lysosome, the tetramer is probably the dominant species. The monomer and tetramer of sulphatase A have similar, or identical, specific activities with nitrocatechol sulphate and 4-methylumbelliferone sulphate as substrates. With nitrocatechol sulphate, sulphatase A shows Michaelis kinetics under conditions where the monomer is the dominant species and non-Michaelis kinetics where the tetramer is dominant. There is apparently a negative cooperativity between the monomer units in the tetramer. In 2 mM sodium taurodeoxycholate and 0.035 M MnCl2, but not in 0.1 M NaCl, the tetramer shows Michaelis kinetics. This is not due to dissociation of the tetramer. The critical micellar concentration of sodium taurodeoxycholate is about 0.8 mM in both 0.1 M NaCl and 0.035 M McCl2 but the aggregation number is greater in the latter.     

Immobilization of lipases on hydrophobic supports involves the open form of the enzyme

The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1 M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength.The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium.     

Assimilatory nitrate reductase from Chlorella. Effect of ionic strength and pH on catalytic activity

Initial velocity studies of Chlorella nitrate reductase showed that increased ionic strength stimulated NADH:nitrate reductase activity by increasing both Vmax and Km for nitrate. Examination of the effect of ionic strength on the various partial activities of nitrate reductase revealed that while NADH:ferricyanide and reduced methyl viologen:nitrate reductase activities were unaffected by ionic strength, NADH:cytochrome c and reduced flavin:nitrate reductase activities were inhibited and stimulated by increased ionic strength, respectively. Comparison of the rates for the partial activities indicated electron transfer from heme to molybdenum to be the rate-limiting step in enzyme turnover. The pH optimum for NADH:nitrate reductase activity was found to be 7.9 while values for the partial activities ranged from 5.5 to 8.1. Phosphate was found to stimulate both NADH:nitrate and reduced methyl viologen:nitrate reductase activities indicating the molybdenum center as the site of interaction.     

Comparative study of solubilized and membrane-bound acetylcholinesterase of sarcolemma]

Comparative kinetic studies of membrane-bound and solubilized sarcolemmal acetylcholinesterase reveal some difference in concentration-activity curves. A deviation from normal Michaelis-Menten kinetics is found in case of membrane-bound acetylcholinesterase. The solubilization of sarcolemma by a solution of high ionic strength or by sonication normalizes the reaction kinetics. It is shown that the amount of SH-groups in intact sarcolemma available for DTNB in the presence of sodium dodecyl sulphate, is about twice of that in the absence of sodium dodecyl sulphate. In case of the solubilized fraction of sarcolemma (by a solution of high ionic strength or by sonication), the amount of SH-groups available for DTNB in the presence and in the absence of sodium dodecyl sulphate is similar.     

Immobilisation and mechanical support of individual protoplasts

Mesophyl cell protoplasts of Vicia faba were suspended in a solution consisting of 10% sodium alginate and 0.4 M mannitol. The protoplasts could be immobilized by cross-linking the alginate in the presence of 100 mM CaCl₂. Changes in the osmolarity of the external medium led to reversible shrinkage and swelling of the entrapped protoplasts. It was demonstrated by using the pressure probe technique that a pressure gradient (cell turgor pressure) of several 100 mbar is built up when the immobilized cells were transferred to hypotonic solution. By complexing the Ca²⁺ in the alginate matrix with sodium citrate buffer the protoplasts could be released from the matrix. No morphological change or alteration of the membrane permeability of the immobilized protoplasts was observed after a storage period of up to 14 days at 4°C in the matrix.     

Sulphur requirements of certain isolates ofAlternaria tenuis Auct

The sulphur nutrition of three isolates ofAlternaria tenuis Auct., isolated from the diseased leaves ofMangifera indica L.,Musa paradisiaca L. andPsidium guajava L., was studied. They were grown on the medium devoid of sulphur as well as on media containing various sources of sulphur viz., ammonium sulphate, sodium hyposulphite, sodium thiosulphate, magnesium sulphate, potassium sulphate, potassium metabisulphite, zinc sulphate and thiourea. Sodium hyposulphite, sodium thiosulphate, magnesium sulphate, potassium sulphate and zinc sulphate were generally found to be satisfactory sources for the growth of all the isolates under study. Poor growth of the different isolates was observed on the medium devoid of sulphur.     

Transient kinetics of electron transfer reactions of flavodoxin: ionic strength dependence of semiquinone oxidation by cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid and computer modeling of reaction complexes

Electron transfer reactions between Clostridum pasteurianum flavodoxin semiquinone and various oxidants [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid (EDTA)] have been studied as a function of ionic strength by using stopped-flow spectrophotometry. The cytochrome c reaction is complicated by the existence of two cytochrome species which react at different rates and whose relative concentrations are ionic strength dependent. Only the faster of these two reactions is considered here. At low ionic strength, complex formation between cytochrome c and flavodoxin is indicated by a leveling off of the pseudo-first-order rate constant at high cytochrome c concentration. This is not observed for either ferricyanide or ferric EDTA. For cytochrome c, the rate and association constants for complex formation were found to increase with decreasing ionic strength, consistent with negative charges on flavodoxin interacting with the positively charged cytochrome electron transfer site. Both ferricyanide and ferric EDTA are negatively charged oxidants, and the rate data respond to ionic strength changes as would be predicted for reactants of the same charge sign. These results demonstrate that electrostatic interactions involving negatively charged groups are important in orienting flavodoxin with respect to oxidants during electron transfer. We have also carried out computer modeling studies of putative complexes of flavodoxin with cytochrome c and ferricyanide, which relate their structural properties to both the observed kinetic behavior and some more general features of physiological electron transfer processes. The results of this study are consistent with the ionic strength behavior described above.     

Evidence for a common genetic regulation of glutamine synthetase and nitrate uptake and reductase in the cyanobacterium Anabaena cycadeae

Summary Nitrate uptake and reductase activities of the cyanobacterium Anabaena cycadeae and its mutant, lacking glutamine synthetase, (the glutamine auxotroph) were measured. The levels of both these enzymes were up to 25-fold higher in the mutant than in the parent (Anabaena cycadeae). the data indicate operation of a common genetic regulatory mechanism controlling the loss of the primary ammonia assimilating enzyme, glutamine synthetase, and derepression of the nitrate uptake and reductase systems.Abbreviations Chl Chlorophyll - GS Glutamine Synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - SDS sodium dodecyl sulphate - Tricine N-tris(hydroxymethyl) methyl glycine - Tris Tris(hydroxymethyl) aminomethane     

Binding of substrates and other anions to yeast phosphoglycerate kinase.

1. The binding of all four substrates to yeast phosphoglycerate kinase has been studied using a gel filtration technique. The binding of phosphate and sulphate anions has also been investigated. 2. Two sites for each adenine nucleotide were found, one site being weaker than the other by between 30 and 50-fold. Only one binding site for the phosphoglycerate substrates was found. 3. 1,3-Bisphosphoglycerate (1,3-P2-glycerate) bound to the enzyme approximately 1000 times tighter than the other three substrates, its dissociation constant being 0.06 micrometer at ionic strength 0.15 M. 4. Sulphate and phosphate were mutually competitive and sulphate competed with the binding of all substrates except MgADP. MgADP bound to the enzyme more weakly in the presence of sulphate. The dissociation constant for sulphate binding was 1.6 mM at ionic strength of 0.15 M, and 0.05 mM at ionic strength 0.015 M. 5. These results are consistent with sulphate acting as a competitive inhibitor, as found by kinetic studies at high sulphate concentrations. The activatory effect of sulphate at lower concentrations and the substrate activation phenomea displayed by this enzyme, are interpreted in terms of a two-step dissociation of 1, 3-P2-glycerate. The presence of moderate concentrations of MgATP, 3-phosphoglycerate or sulphate causes acceleration of the rate of dissociation of the product, 1, 3-P2-glycerate, this being the rate-limiting step in the overall enzyme reaction.     

Use of silica-immobilized humin for heavy metal removal from aqueous solution under flow conditions

Humin extracted from Sphagnum peat moss was immobilized in a silica matrix and column experiments were performed in order to evaluate the removal and recovery of metal ions from aqueous solution under flow conditions. These experiments also allowed testing the recycling capacity of the column. Single-element solutions of Cu(II) and Pb(II), and a multi-metal solution containing Cd(II), Cu(II), Pb(II), Ni(II), and Cr(III) were passed through the columns at a flow rate of 2 ml/min. A 0.5 M sodium citrate solution was used as the stripping agent in the metal-ion recovery process. Humin immobilized in the silica matrix exhibited a similar, and in some cases, even a higher capacity than other biosorbents for the removal of metal ions from aqueous solutions under flow conditions. The sodium citrate was effective in removing Cu(II), Pb(II), Cd(II), and Ni(II) from the metal saturated column. The selectivity of the immobilized biomass was as follows: Cr(III)>Pb(II)>Cu(II)>Cd(II)>Ni(II). This investigation provides a new, environmentally friendly and cost-effective possibility to clean up heavy-metal contaminated wastewaters by using the new silica-immobilized humin material.     

Electrochemical evaluation of lactate dehydrogenase immobilized in polyacrylamide gels

Lactate dehydrogenase (EC 1.1.1.27) has been immobilized in polyacrylamide gels over a platinum grid matrix. The immobilized enzyme is used to oxidize L-lactate in the presence of nicotinamide adenine dinucleotide (NAD+) and ferricyanide. The NADH produced is then chemically oxidized back to NAD+ by ferricyanide. The coupled reduction of ferricyanide ions to ferrocyanide ions results in a measurable electrochemical potential. This measurable zero-current potential is found to be Nernstian in nature and directly proportional to the logarithm values of L-lactate concentration over the range of 2 X 10(-5) to 5 X 10(-2)M. The results indicate that immobilized lactate dehydrogenase can be incorporated into a system to detect L-lactate acid in aqueous solutions.     

Characteristics and applications of adsorbents for pyrogen removal

Characteristics and applications of immobilized histidine and immobilized histamine for pyrogen removal were investigated. Immobilized histidine showed a high affinity for pyrogen at low ionic strength and over a wide pH range. The adsorption capacity was 0.53 mg of lipopolysaccharide per milliliter of the adsorbent. The apparent dissociation constant was 1.57 X 10(-9) M. The adsorption of pyrogen to immobilized histidine decreased with increasing ionic strength, but pyrogen could be adsorbed even at ionic strengths of gamma/2 = 0.05-0.1, at which other substances were little adsorbed; that is, specific adsorption of pyrogen was observed. The adsorption of pyrogen could be increased at ionic strengths of gamma/2 = 0.05-0.1 by using a lower flow rate or a longer column length. Immobilized histidine and immobilized histamine could be used for the removal of natural pyrogens contaminating various useful low-molecular-weight compounds as well as high-molecular-weight compounds such as proteins.     

Association of myelin basic protein with detergent micelles.

Equilibrium measurements of the binding of central nervous system myelin basic protein to sodium dodecyl sulphate, sodium deoxycholate and lysophosphatidylcholine have been obtained by gel permeation chromatography and dialysis. This protein associates with large amounts of each of these surfactants: the apparent saturation weight ratios (surfactant/protein) being 3.58 +/- 0.12 and 2.30 +/- 0.15 for dodecyl sulphate at ionic strengths 0.30 and 0.10, respectively 1.34 +/- 0.10 for deoxycholate (at 0.12 ionic strength) and 4.0 +/- 0.5 for lysophosphatidylcholine. Binding to the ionic surfactants increases markedly close to their critical micelle concentrations. Sedimentation analysis shows that at 0.30 ionic strenght in excess dodecyl sulphate the protein is monomeric. It becomes dimeric when the binding ratio falls below 1 at a free detergent concentration of approximately 0.25 mM: below this concentration much of the protein and deterent forms an insoluble complex. The amount of dodecyl sulphate bound at high concentrations and at both above-mentioned ionic strengths corresponds closely to that expected for interaction of a single poly-peptide with two micelles. Variability of deoxycholate micelle size on interaction with other molecules precludes a similar analysis for this surfactant. Association was observed only with single micelles of lysophosphatidylcholine. The results provide strong evidence for dual lipid-binding sites on basic protein and indicate that lipid bilayer cross-linking by this protein may be effected by single molecules.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrate-ferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the 'association' rate but also by increasing the 'dissociation' rate for bound cytochrome c converting the 'primary' (T) site at high salt concentrations into a site similar kinetically to the 'secondary' (L) site at low ionic strength. A finite Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.