A special role of phosphate in the stability of lactate dehydrogenase against destruction by a polyelectrolyte

It has been shown by X-ray structure analysis that proteins have specific anion-binding sites for sulfate, citrate, and phosphate ions; however, the functional role of these anions is not always clear. Thus, it is unknown which of two phosphate anions, mono- or divalent, determines the stability of cellular proteins under stress conditions. In the present work, the influence of phosphate, sulfate, and chloride on the stability of lactate dehydrogenase (LDH) in the presence of poly(styrenesulfonate) (PSS) has been investigated by the methods of steady-state kinetics and intrinsic protein fluorescence. The study is based on the analysis of differences between the influence of phosphate and sulfate ions on the process at two pH values, 6.2 and 7.0, at which the ratio of the concentrations of mono- and bivalent phosphate forms differs, whereas sulfate remains in the bivalent form. It was shown that the differences between the influence of phosphate and sulfate ions at pH 7.0 were greater; divalent phosphate ions much more effectively stabilized LDH against destruction by a polyelectrolyte compared with sulfate and monovalent phosphate. It was concluded that, of two anion-binding sites of the LDH molecule, the intersubunit center plays the most important role in its stabilization against destruction by polyelectrolyte, and, of two forms of phosphate anions, its bivalent form HPO ₄ ^?2 plays the stabilizing role.     

Influence of glycerol on the structure and redox properties of horse heart cytochrome c. A circular dichroism and electrochemical study

The effect of glycerol on the structure and redox properties of horse heart cytochrome c was investigated by absorption spectroscopy, circular dichroism, and dc cyclic voltammetry techniques. The results show that the organic solvent increases the -helix structure of the protein and induces slight changes at the active-site environment; however, the overall tertiary structure does not appear to be significantly perturbed. Glycerol stabilizes cytochrome c, the free energy of denaturation (G ₀) being approximately 0.7 kcal/mol larger than that determined in phosphate buffer under the same conditions, and influences the heterogeneous electron transfer kinetics at a chemically modified gold electrode; on the other hand, the redox potential of the protein is unaltered. On the whole, the results obtained indicate that glycerol acts as a suitable stabilizing agent of cytochrome c, which is of interest for application in biotechnology; the organic solvent does not alter the tertiary structure significantly or the redox properties of the protein. This has to be interpreted not only in terms of the glycerol-induced solvent ordering around the protein surface, but also as due to the specific features of the protein matrix.     

Ethanol dissimilation in Desulfovibrio

During growth of ethanol plus sulfate Desulfovibrio gigas and three other Desulfovibrio strains tested contained high NAD-dependent alcohol dehydrogenase activities and dye-linked aldehyde dehydrogenase activities. In lactate-grown cells these activities were lower or absent. In D. gigas an NADH dehydrogenase activity was found which was higher during growth on ethanol than during growth on lactate. The NADH dehydrogenase activity appeared to consist of at least three different soluble enzymes. The aldehyde dehydrogenase activity in D. gigas was highest with benzylviologen as an acceptor and was strongly stimulated by potassium ions. Coenzyme A or phosphate dependency could not be shown, indicating that acetyl-CoA or acetyl phosphate are not intermediates in the conversion of acetaldehyde to acetate.In the absence of sulfate D. gigas was able to convert ethanol to acetate by means of interspecies hydrogen transfer to a methanogen. This conversion, however, did not lead to growth of the Desulfovibrio.Abbreviations DH dehydrogenase - BV²⁺/BV⁺ oxidized/reduced benzylviologen - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide - MV²⁺/MV⁺ oxidized/reduced methylviologen - PMS phenazine methosulfate     

Thermostabilization of Ovotransferrin by Anions for Pasteurization of Liquid Egg White

The effects of anions on the thermostability of ovotransferrin (oTf) were investigated. The temperature, T_m, causing aggregation of oTf was measured in the presence or absence of anions, and the denaturation temperature, T_m^DSC, was also determined by differential scanning calorimetry (DSC) in the presence of the citrate anion. We found that some anions (phosphate, sulfate and citrate) raised temperature T_m of oTf by about 5–7 °C. However, neither sodium chloride nor sodium bicarbonate raised T_m by that much. Temperature T_m was increased by increasing the concentration of the citrate anion, and was in good agreement with denaturation temperature T_m^DSC, suggesting that denaturation of the oTf molecules resulted in aggregation of oTf. We also demonstrated that the anions, especially sulfate, repressed the heat-aggregation of liquid egg white.

The Van’t Hoff plot from the T_m and ΔH_d values revealed that two anion-binding sites were concerned with heat stabilization. These binding sites may have been concerned with sulfate binding (not bicarbonate binding) that is found in the crystal structure of apo-form of oTf, since the bicarbonate anion did not raise T_m.     

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

Effect of salts on the activity of carrot phosphofructokinase

Phosphofructokinase (EC 2.7.1.11) from carrot roots was activated by a number of salts. Increase in salt concentration beyond the optimum generally led to a decrease in enzyme activity. Salts of the multivalent anions sulfate and phosphate were very effective activators and inhibitors. Potassium acetate and potassium succinate were also activators. Potassium tartrate and potassium citrate produced a small stimulation at low concentration but with further increase they became inhibitory. The results suggested that the salt effect was largely due to anions rather than cations. Salts such as NaCl, KCl, and in particular potassium phosphate, relieved the inhibition of carrot phosphofructokinase by phosphoenolpyruvate. KCl and potassium phosphate also reversed the inhibition of carrot phosphofructokinase by citrate. The possible significance of these observations in the regulation of glycolysis and carbohydrate metabolism, and in salt respiration is discussed.     

Selective stabilization of a labile mutant form of bovine pancreatic ribonuclease A by antibodies

The region between the amino acids 31-46 was previously identified as being first exposed during thermal unfolding of bovine pancreatic ribonuclease A (RNase). The exchange of one amino acid (Leu35toSer) in this unfolded region of RNase is shown to have a dramatic destabilizing effect (T_m=9 °C). Antibodies raised against a peptide corresponding to the sequence of the labile region, S32-V43, of RNase were effective in stabilizing L35S-RNase against thermal inactivation (65 °C for 2 h) and surpassed the stabilization effect of antiRNase antibodies. An 11% contribution to the stabilizing effect of antiRNase antibodies resulted from antibodies recognizing the unfolding region of the enzyme.     

Inhibition of the phosphate self-exchange flux in human erythrocytes and erythrocyte ghosts

Summary The phosphate self-exchange flux in resealed erythrocyte ghosts and in amphotericin B (5.5 m) permeabilized erythrocytes has been studied. The phosphate self-exchange flux exhibits an S-shaped concentration dependence and a self-inhibition in permeabilized red cells while in erythrocyte ghosts no self-inhibition of the phosphate flux has been observed. The apparent halfsaturation constants and the apparent Hill coefficients were assessed by the double reciprocal Hill plots of versus 1/[P] ⁿ . The phosphate half-saturation constants amount to approx. 125mm in ghosts and to about 75mm in permeabilized cells while the apparent Hill coefficients amount to 1.15 and to 1.65 (pH 7.2, 25°C), respectively. Both chloride and sulfate elicit a mixed-type inhibition of the phosphate self-exchange flux. In permeabilized cells, chloride and sulfate shift the flux optimum towards higher phosphate concentrations and reduce the apparent Hill coefficients. In erythrocyte ghosts, the apparent Hill coefficients are insensitive to these anions. The double reciprocal Hill plots indicate a mixed-type inhibition of the phosphate self-exchange flux by DNDS, salicylate and dipyridamole and a noncompetitive inhibition of the phosphate self-exchange flux by phlorhizin. By contrast, the Hill-Dixon plots for chloride and sulfate indicate a competitive inhibition of the phosphate self-exchange flux in erythrocyte ghosts and a mixed-type inhibition in permeabilized cells and provide Hill coefficients of greater than unity for chloride and sulfate. The Dixon plots for DNDS, salicylate, phlorhizin and dipyridamole show a noncompetitive inhibition of the phosphate flux and provide apparent Hill coefficients of 0.95–1.0 for inhibitor binding. Using the Debye-Hückel theory, the effects of ionic strength upon phosphate transport and inhibitor binding can be eliminated. The results of our studies provide strong evidence for the assumption that electrostatic forces are involved in phosphate transport and in inhibitor binding.     

Sulfate activation in Desulfotomaculum

Enzyme activities conceivably involved in the activation of sulfate were studied with Desulfotomaculum ruminis, D. acetoxidans, D. nigrificans, D. orientis, and Desulfovibrio vulgaris. Cell lysates of these species revealed activities of at least 8 nkat/mg protein (i.e., 480 nmol per min and mg protein) of ATP sulfurylase, acetate kinase, phosphotransacetylase and adenylate kinase. ADP sulfurylase was not detected. Pyrophosphatase activity was high (73 to 97 nkat/mg protein) in Desulfotomaculum orientis and Desulfovibrio vulgaris. In these strains pyrophosphatase was activated by addition of a reductant (dithionite). In Desulfotomaculum ruminis, D. acetoxidans, and D. nigrificans, only low pyrophosphatase activity (2.5 to 6.3 nkat/mg protein) was measured, which was not reductant-activated. Some hints indicated a membrane association of the pyrophosphatase in D. ruminis, and possibly also in D. acetoxidans and D. nigrificans. Activities of a pyrophosphate-dependent acetate kinase (PP_i:acetate kinase), a PP_i:AMP kinase or a polyphosphate:AMP kinase were not detected or negligible. The results are not in favour of the assumption that pyrophosphate formed by ATP sulfurylase during sulfate activation might be utilized to form acetyl phosphate in Desulfotomaculum species. Contrary results of other authors were shown to be artefacts caused by chemical hydrolysis of acetyl phosphate in the molybdate-sulfuric acid reagent used for phosphate determination.Abbreviations P_i orthophosphate - PP_i pyrophosphate - APS adenosine phosphosulfate - AP₅A, P¹ P⁵-di(adenosine-5-)pentaphosphate - CTAB cetyltrimethylammonium bromide - MOPS 3-(N-morpholino)propanesulfonic acid - HEPES N(-2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid     

Production of Yeast Cells from Methanol

Using immunochemical technique thermal denaturation of soybean 11S globulin, dissolved in different ionic strength solutions (µ=0~4.0) and heated at 100°C for 5 min, has been quantitatively studied. The curves of the percentage of antigenicity remaining were obtained as a function of salt concentration. The 11S globulin became strongly resistant to thermal denaturation with increasing both KCl and potassium phosphate. The stabilizing effect (in terms of percent antigenicity) was separated into three regions. At ionic strength below 0.7, potassium phosphate had no stabilizing effect while KCl had aslightly effect. The rise in stabilizing effect up to about 50%, near 1.0~1.5 µ, represented a second transition to a different denatured state which retains undissociated molecule. At rises up to 75~95%, near 2.5~3.5µ, a different conformational state resulted in which thermally denatured 11S globulin maintained almost intact native conformation after heating. The selection of an adequate ionic strength of protein solution has enabled preparation of thermally denatured 11S globulins which have desired-residual amounts of structured regions.     

Thermostabilization of ovotransferrin by anions for pasteurization of liquid egg white

The effects of anions on the thermostability of ovotransferrin (oTf) were investigated. The temperature, T(m), causing aggregation of oTf was measured in the presence or absence of anions, and the denaturation temperature, T(m)(DSC), was also determined by differential scanning calorimetry (DSC) in the presence of the citrate anion. We found that some anions (phosphate, sulfate and citrate) raised temperature T(m) of oTf by about 5-7 degrees C. However, neither sodium chloride nor sodium bicarbonate raised T(m) by that much. Temperature T(m) was increased by increasing the concentration of the citrate anion, and was in good agreement with denaturation temperature T(m)(DSC), suggesting that denaturation of the oTf molecules resulted in aggregation of oTf. We also demonstrated that the anions, especially sulfate, repressed the heat-aggregation of liquid egg white.The Van't Hoff plot from the T(m) and DeltaH(d) values revealed that two anion-binding sites were concerned with heat stabilization. These binding sites may have been concerned with sulfate binding (not bicarbonate binding) that is found in the crystal structure of apo-form of oTf, since the bicarbonate anion did not raise T(m).     

Kinetic characteristics of the sulfate self-exchange in human red blood cells and red blood cell ghosts

Summary The sulfate and the chloride self-exchange fluxes were determined by measuring the rate of the tracer efflux from radioactively labeled human red blood cells and red blood cell ghosts. The concentration dependence and the pH-dependence of the sulfate self-exchange flux were studied. In addition, the effects of some monovalent and divalent anions on the sulfate and the chloride self-exchange fluxes were investigated.The sulfate self-exchange fluxes saturate, exhibiting a concentration maximum at sulfate concentrations between 100 and 300mm (25°C). The position of the concentration maximum depends upon pH. At high sulfate concentrations a self-inhibition of the flux becomes apparent. The apparent half-saturation constant and the apparent self-inhibition constant at pH 7.2 were 30mm and 400mm respectively. Within the pH range of 6.3–8.5, both constants decreased with increasing pH. No saturation of the sulfate self-exchange flux was observed if the sulfate concentration was raised by substituting sulfate for isoosmotic amounts of a second salt (NaCl, NaNO₃, Na-acetate, Na-lactate, Na-succinate or Na₂HPO₄). Red blood cells and red blood cell ghosts display the same pattern of concentration responsiveness.The sulfate self-exchange flux exhibits a pH-maximum at about pH 6.2 (37°C). The location of the pH-maximum is little affected by variations of the sulfate concentration. The logarithmic plots (log vs. pH) revealed that the flux/pH relation can be approximated by two straight lines. The slopes of the alkaline branches of the flux/pH curves range from –0.55 to –0.86, the slopes of the branches of the curves range from 0.08 to 1.14 and were strongly affected by changes of the sulfate concentrations. The apparent pK's obtained from the alkaline and from the acidic branches of the flux/pH curves were about 7.0 and 6.0, respectively. Intact red blood cells and red blood cell ghosts display the same type of pH-dependency of the sulfate self-exchange flux.The sulfate self-exchange flux is competitively inhibited by nitrate, chloride, acetate, oxalate and phosphate. The chloride self-exchange flux is competitively inhibited by thiocyanate, nitrate, sulfate and phosphate. The inhibition constants for the various anion species increase in the given sequence.The results of our studies indicate that the sulfate self-exchange flux is mediated by a two-site transport mechanism consisting either of a mobile carrier or a two-site pore. The experiments reported in this paper do not permit distinguishing between both transport mechanisms. The similarities of the sulfate and the chloride self-exchange flux and the mutual competition between sulfate and chloride point to a common transport system for both anion species.     

Indirect photometric ion chromatographic analysis of anions in Haematococcus pluvialis culture media

An indirect photometric ion chromatographic method for the simultaneous determination of chloride, nitrate and sulfate ions was developed and applied to the determination of anions, mainly nitrate, in the alga Haematococcus pluvialis culture media. Using phthalic acid/sodium tetraborate aqueous solution as the mobile phase, anions can be detected indirectly by a UV detector. The calibration curves for these anions gave good linearity from 1 to 1000 g ml^–1.     

Sulfate and sulfite translocation via the phosphate translocator of the inner envelope membrane of chloroplasts

The permeability of the inner envelope membranes of spinach (Spinacia oleracea) chloroplasts to sulfite and sulfate was investigated in vitro, using the technique of silicone oil centrifugal filtration. The results show that there is a permeability towards both ions, resulting in rates of uptake of about 1.0 (SO ₃ ^2- ) and 0.7 (SO ₄ ^2- ) mol mg chlorophyll^-1 h^-1 respectively (external concentration 2 mmol l^-1). The rates depend on the external concentration of the anions. Anion exchange experiments with ³⁵S-preloaded chloroplasts indicate that sulfite and sulfate are exchanged for inorganic phosphate, phosphoglyceric acid, and dihydroxyacetone phosphate with rates up to 14 nmol mg chlorophyll^-1 min^-1. There is no exchange for glucose-6-phosphate and malate. Because of the similarities to the transport of inorganic phosphate and triose phosphates the results give evidence that the phosphate translocator of the inner envelope membrane of chloroplasts is also involved in sulfite and sulfate transport — at least in part.Abbreviations DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - P_i inorganic phosphate - S_i sultite, sulfate     

Association-dissociation and denaturation-renaturation of high-molecular-weight protein: carmin from safflower seed (Carthamus tinctorius L.) in alkaline solution

The effect of alkalinepH on the association, dissociation, and denaturation of carmin, the high-molecular-weight protein from safflower seed was investigated in thepH range 7–12, using various biophysical techniques. The results indicate that the multimeric protein carmin dissociates atpH 8.0 where denaturation has not set in. The association-dissociation of the protein can be represented schematically as 11S 7S 4S 2S. AbovepH 10, the protein undergoes simultaneous dissociation and denaturation. The denaturation process appears to be complete at pH 12.5. The protein undergoes conformational change and covalent modifications and cleavage during the denaturation process. A reversibility study shows that the process of dissociation is reversible to a large extent, whereas denaturation appears to be irreversible. These results are discussed in terms of association-dissociation, denaturation and alkaline-catalyzed covalent modifications and cleavage of seed proteins.     

Import of a new chloroplast inner envelope protein is greatly stimulated by potassium phosphate

A cDNA clone encoding a major chloroplast inner envelope membrane protein of 96 kDa (IEP96) was isolated and characterized. The protein is synthesized as a larger-molecular-weight precursor (pIEP96) which contains a cleavable N-terminal transit sequence of 50 amino acids. The transit peptide exhibits typical stromal targeting information. It is cleaved in vitro by the stromal processing peptidase, though the mature protein is clearly localized in the inner envelope membrane. Translocation of pIEP96 into chloroplasts is greatly stimulated in the presence of 80 mM potassium phosphate which results in an import efficiency of about 90%. This effect is specific for potassium and phosphate, but cannot be ascribed to a membrane potential across the inner envelope membrane. Protein sequence analysis reveals five stretches of repeats of 26 amino acids in length. The N-terminal 300 amino acids are 45% identical (76% similarity) to the 35 kDa -subunit of acetyl-CoA carboxyl-transferase from Escherichia coli. The C-terminal 500 amino acids share significant similarity (69%) with USOI, a component of the cytoskeleton in yeast.Abbreviations P_i phosphate - IEP inner envelope membrane protein - pIEP precursor form of IEP - SSU small subunit of ribulose-1,5-bisphosphate carboxylase oxygenase - IEP96_pep peptide specific antiserum to IEP96 - IEP96_pol polyspecific antiserum to IEP96     

Contractile eukaryotic flagella: Centrin is involved

Summary Isolated transverse flagella ofPeridinium inconspicuum (Dinophyceae) undergo a rapid Ca²⁺-induced (50M Ca²⁺) contraction in the absence of exogenous ATP. Longitudinal flagella from the same species do not contract under these conditions. Contraction leads to a supercoiling of the axoneme and a shortening of the paraxonemal fiber that accompanies the axoneme over most of its length. Using a polyclonal antibody generated against centrin, a 20 kDa Ca²⁺-modulated contractile protein of striated flagellar roots of the green flagellateTetraselmis striata, we have found that the paraxonemal fiber in transverse flagella of three taxa ofDinophyceae is immunoreactive by indirect immunofluorescence. The localization of the antigen in the paraxonemal fiber of transverse flagella was confirmed by two-colour double immunofluorescence using monoclonal mouse-anti--tubulin for identification of the axoneme. No structure was immunoreactive to anticentrin in the longitudinal flagella of all taxa. Electrophoretic and immunoblot analysis of isolated flagella ofP. inconspicuum show that the antigen is a 21 kDa protein, indicating that it is either centrin or a closely related protein. We conclude that centrin confers contractility to the transverse flagellum of dinoflagellates and possibly to other contractile eukaryotic flagella.Abbreviations ASP-H artificial seawater medium with Hepes-buffer - BSA bovine serum albumine - DTT dithiothreitol - EGTA ethylene glycol bis(2-amino-ethylether)tetraacetic acid - FITC fluorescein isothiocyanate - MT buffer microtubule stabilizing buffer - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - TLCK N-p-tosyl-l-lysine chloromethyl ketone - TRITC tetramethylrhodamine isothiocyanate     

Approaches for increasing the solution stability of proteins

Stabilization of proteins through proper formulation is an important challenge for the pharmaceutical industry. Two approaches for stabilization of proteins in solution are discussed. First, work describing the effect of additives on the thermally induced denaturation and aggregation of low molecular weight urokinase is presented. The effects of these additives can be explained by preferential exclusion of the solute from the protein, leading to increased thermal stability with respect to denaturation. Diminished denaturation leads to reduced levels of aggregation. The second approach involves stoichiometric replacement of polar counter ions (e.g., chloride, acetate, etc.) with anionic detergents, in a process termed hydrophobic ion pairing (HIP). The HIP complexes of proteins have increased solubility in organic solvents. In these organic solvents, where the water content is limited, the thermal denautration temperatures greatly exceed those observed in aqueous solution. In addition, it is possible to use HIP to selectively precipitate basic proteins from formulations that contain large amounts of stabilizers, such as human serum albumin (HSA), with a selectivity greater than 2000-fold. This has been demonstrated for various mixtures of HSA and interleukin-4. (c) 1995 John Wiley & Sons, Inc.     

Analysis of DNA–Protein Contacts in a Complex between Methyltransferase SsoII and a Promoter Region of the SsoII Restriction–Modification Genes

Heterocyclic bases and phosphate groups involved in the DNA–methyltransferase SsoII (M·SsoII) interaction were identified in the regulatory DNA region localized within the promoter region of the SsoII restriction–modification genes by footprinting with the use of formic acid, hydrazine, dimethyl sulfate, and N-ethyl-N-nitrosourea as modifying agents. It has been established that the enzyme interacts with three guanines, one adenine, two thymines, and three phosphate groups of each strand of the DNA duplex. These heterocyclic bases and phosphate groups are disposed symmetrically within the 15-mer inverted repeat of the regulatory DNA region. It has been demonstrated by footprinting with dimethyl sulfate that the C7 atoms of guanines interacting with the enzyme are exposed to the DNA major groove. Two theoretical models were built describing the contacts in a complex between M·SsoII and the regulatory DNA region.