Effects of anions on the activation thermodynamics and fluorescence emission spectrum of alkaline phosphatase: evidence for enzyme hydration changes during catalysis

The effect of anions on the thermodynamic activation functions for a model enzyme, calf intestinal alkaline phosphatase (EC 3.1.3.1), have been studied in order to examine the role of protein hydration changes in establishing the energetics of enzyme catalysis. The influences of these anions on the activation volume (delta V) and activation free energy (delta G) reflected clear Hofmeister (lyotropic) series effects, in the order F- greater than Cl- greater than Br- greater than I- (order of increasing salting-out potential). A pronounced covariation was observed between the influences of these anions on Vmax, which is proportional to delta G, and on the negative activation volume of the reaction. Fluoride was able to counteract the influences of Br- and I- on both Vmax and delta V when combinations of these anions were employed. The effects of Br- and I- on Vmax and delta V were more pronounced at lower temperatures. The control delta V was increasingly negative at reduced temperatures. The effects of the neutral salts and propanol on delta V and delta G, as well as the effects of salting-in anions on the activation enthalpy and the negative activation entropy of the reaction, are consistent with a model which proposes that peptide groups or polar side chains on the native enzyme exergonically increase their exposure to solvent during the catalytic activation event. These conclusions are in accord with the known free energy, enthalpy, entropy, and volume changes which occur when model peptide groups are transferred between water and concentrated salt solutions. Consistent with the kinetic results, the fluorescence emission wavelength maximum of alkaline phosphatase increased in the presence of anions in the order F- greater than Cl- greater than Br- greater than I-. The salting-out ion (F-) and the salting-in ions (Br- and I-) shifted lambda max in different directions, and these lambda max shifts could be counterbalanced by using equimolar combinations of salting-in and salting-out anions. Control experiments with a model compound, N-acetyltryptophanamide, showed that the spectra shifts caused by the salts did not result solely from differential quenching by the anions of the solvent-exposed tryptophan(s) on the enzyme. Hofmeister additivity phenomena indicated that the solvent is at the basis of these salt-induced enzyme structural changes. It is concluded that changes in protein solvation during enzymic reactions contribute significantly to the thermodynamic activation parameters in both the native and the salt-perturbed enzyme.     

Anion binding properties of human serum albumin from halide ion quadrupole relaxation.

The nuclear magnetic quadrupole relaxation enhancement of 35Cl-, 81Br-, and 12I- anions on binding to human serum albumin has been studied under conditions of variable protein and anion concentration and also in the presence of simple inorganic, amphiphilic, and complex anions which compete with the halide ions for the protein anion binding sites. Two classes of anion binding sites with greatly different binding constans were identified. Experiments at variable halide ion concentration were employed to determin the Cl- and I- binding constants. By means of 35 Cl nuclear magnetic resonance (NMR) the relative affinity for different anions was determined by competition experiments for both the strong and the weak anion binding sites. Anion binding follows the sequence SO42- smaller than F- smaller than CH3COO- smaller than Ci- smaller Br- smaller than NO3- smaller than I- smaller than ClO4- smaller than SCN- smaller than Pt(CN)42- smaller than Au(CN)2- smaller than CH3(CH2)11OSO3- for the high affinity sites, and the sequence SO42- congruent to F- congruent to Cl- smaller CH3COO- smaller than NO3- smaller than Br- smaller than I- smaller than ClO4- smaller than SCN- for the low affinity sites. These series are nearly identical with the well-known lyotropic series. Consequently, those effects of anions on proteins described by the lyotropic series can be correlated with the affinities of the anions for binding to the protein. The data suggest that the physical nature of the interaction is the same for both types of biding sites, and that the differences in affinity between different binding sites must be explained in terms of tertiary structure. Analogous experiments performed using 127I- quadrupole relaxation gave results very similar to those obtained with 35Cl-. A comparison between the Cl-, Br- and I- ions revealed that, as a result of the increasing affinity for the weak anion binding sites in the series Cl- smaller than Br- smaller than I-, Cl- is much more useful as a probe for the specific anion binding sites than the other two halide ions. The findings with human serum albumin in this and other respects are probably of general relevance in studies of protein-anion interactions. In addition to competition experiments, the magnitude of the relaxation rate is also discussed. Line broadening not related to anion binding to the protein is found to be small. A comparison of transverse and longitudinal 35Cl relaxation rates gives a value for the quadrupole coupling constant of the high affinity sites in good agreement with a calculated coupling constant assuming anion binding to arginine.     

Lysis of Streptococcus mutans by hen egg white lysozyme and inorganic sodium salts.

Streptococcus mutans BHT was grown in a synthetic medium containing radioactive thymidine to monitor deoxyribonucleic acid release. Kinetic experiments demonstrated that although lysozyme alone could not liberate deoxyribonucleic acid, cellular deoxyribonucleic acid was liberated from lysozyme-treated cells by addition of low concentrations of inorganic sodium salts. When the salts were tested for their ability to dislodge cell-bound tritiated lysozyme, the extent of the initial release of enzyme by individual anions correlated with the anion potency for deoxyribonucleic acid liberation (SCN- greater than ClO4- greater than I- greater than Br- greater than NO3- greater than Cl- greater than F-), although the total amount of lysozyme dislodged did not correspond directly with cell lysis. Differences in the effectiveness of anions (SCN-, HCO3-, Cl- and F-) in potentiating cell lysis could be enhanced or minimized by varying the lysozyme, anion, and bacterial cell concentrations. As the anion concentration was increased for each enzyme concentration and cell concentration, the lysis increased, in some cases markedly, until maximum levels of released deoxyribonucleic acid were attained. The maximum levels of lysis of SCN- and HCO3- were similar and were greater than those for Cl- and F-. In addition, the maximum levels were observed to increase for each of the anions as the concentration of lysozyme increased.     

Anion recognition by thiostrepton

A bicyclic polypeptide antibiotic thiostrepton forms both 1:1 and 1:2 complexes with anions (as tetrabutylammonium salts) in organic solvents with K2 >K1 for F- and K2Cl-, Br-, HSO4-, H2PO4-, but in CHCl3 they follow a different order: Cl- approximatelyHSO4- >F- approximately AcO- > Br > H2PO4-. The binding mode of anions to thiostrepton is discussed on the basis of solvent effects on the complexation selectivity.     

Interactions of bromide, iodide, and fluoride with the pathways of chloride transport and diffusion in human neutrophils

Isolated human neutrophils possess three distinct pathways by which Cl- crosses the plasma membrane of steady state cells: anion exchange, active transport, and electrodiffusion. The purpose of the present work was to investigate the selectivity of each of these separate processes with respect to other external halide ions. (a) The bulk of total anion movements represents transport through an electrically silent anion-exchange mechanism that is insensitive to disulfonic stilbenes, but which can be competitively inhibited by alpha-cyano-4-hydroxycinnamate (CHC; Ki approximately 0.3 mM). The affinity of the external translocation site of the carrier for each of the different anions was determined (i) from substrate competition between Cl- and either Br-, F-, or I-, (ii) from trans stimulation of 36Cl- efflux as a function of the external concentrations of these anions, (iii) from changes in the apparent Ki for CHC depending on the nature of the replacement anion in the bathing medium, and (iv) from activation of 82Br- and 125I- influxes by their respective ions. Each was bound and transported at roughly similar rates (Vmax values all 1.0-1.4 meq/liter cell water.min); the order of decreasing affinities is Cl- greater than Br- greater than F- greater than I- (true Km values of 5, 9, 23, and 44 mM, respectively). These anions undergo 1:1 countertransport for internal Cl-. (b) There is a minor component of total Cl- influx that constitutes an active inward transport system for the intracellular accumulation of Cl- [( Cl-]i approximately 80 meq/liter cell water), fourfold higher than expected for passive distribution. This uptake is sensitive to intracellular ATP depletion by 2-deoxy-D-glucose and can be inhibited by furosemide, ethacrynic acid, and CHC, which also blocks anion exchange. This active Cl- uptake process binds and transports other members of the halide series in the sequence Cl- greater than Br- greater than I- greater than F- (Km values of 5, 8, 15, and 41 mM, respectively). (c) Electrodiffusive fluxes are small. CHC-resistant 82Br- and 125I- influxes behave as passive leak fluxes through low-conductance ion channels: they are nonsaturable and strongly voltage dependent. These anions permeate the putative Cl- channel in the sequence I- greater than Br- greater than Cl- with relative permeability ratios of 2.2:1.4:1, respectively, where PCl approximately 5 X 10(-9) cm/s.     

Lyotropic anions. Na channel gating and Ca electrode response

The effects of external anions on gating of Na channels of frog skeletal muscle were studied under voltage clamp. Anions reversibly shift the voltage dependence of peak sodium permeability and of steady state sodium inactivation towards more negative potentials in the sequence: methanesulfonate less than or equal to Cl- less than or equal to acetate less than Br- less than or equal to NO-3 less than or equal to SO2-4 less than benzenesulfonate less than SCN- less than ClO-4; approximately the lyotropic sequence. Voltage shifts are graded with mole fraction in mixtures and are roughly additive to calcium shifts. The peak PNa is not greatly affected. Except for SO2-4, these anions did not change the Ca++ activity of the solutions as measured with the dye murexide. Shifts of gating can be explained as the electrostatic effect of anion adsorption to the Na channel or to nearby lipid. Such adsorption is expected to follow the lyotropic series. Anions also interfere significantly with the response of a Ca-sensitive membrane electrode following the same sequence of effectiveness as the shifts of gating. The lyotropic anions decrease the Ca++ sensitivity and cause anomalously negative responses of the Ca electrode because these anions are somewhat permeant in the hydrophobic detector membrane.     

The effect of Cl- depletion and X- reconstitution on the oxygen-evolution rate, the yield of the multiline manganese EPR signal and EPR signal II in the isolated Photosystem-II complex

The role of Cl- in photosynthetic O2 evolution has been investigated by measurement of the steady-state O2 rate and EPR of the electron donors responsible for the S2 multiline signal and Signal IIs upon Cl- depletion and substitution in Photosystem II membranes. Cl- removal has three effects upon the donor side of Photosystem II. (1) It abolishes O2 evolution reversibly, while decreasing the yield of the S2 multiline signal indicative of the manganese site of the O2-evolving complex in the S2 oxidation state. This decrease is brought about by (2) the reversible disconnection of the manganese complex from the reaction center; and by (3) deactivation of S1 centers having reduced primary acceptor QA to form SO centers having a reduced Signal IIs species. Reactivation of O2 evolution by anions confirms earlier work showing a requirement for a univalent anion of optimum charge density. The observed order of reactivation is Cl- greater than Br- approximately NO3- much greater than OH- approximately F-. Reactivation of the S2 multiline signal follows Cl- approximately Br- greater than NO3- approximately OH- greater than F-, in near correspondence with reactivation of O2-evolution rates. Cl- titrations of F- -inhibited samples reveal two binding sites for Cl- which differ in binding affinity by 11-fold. The higher-affinity site reactivates the S1----S2 light reaction, while the lower-affinity site reactivates the S3----S0 light reaction. The high affinity site is located within the O2-evolving complex at an undetermined site, while the lower-affinity site functions in coupling the reaction center photochemistry to the O2-evolving complex. The results are compared with Cl-/F- exchange equilibria for Mn3+ in solution. A model for the lower S-state transitions is presented in which specific oxidation state assignments are made for some of the donors and acceptors of Photosystem II.     

Light-scattering investigations of the subunit dissociation of human hemoglobin A. Effects of various neutral salts

The effectiveness of various salts of the Hofmeister series as dissociating agents for human hemoglobin A tetramers has been investigated by light-scattering molecular-weight measurements. Dissociation of hemoglobin to half-molecules of alpha beta dimers follows the order of the series dictated predominantly by the sequence of the anions F- less than Cl- less than Br- less than ClO4- less than SCN-, I-, with the cations Na+ and K+ having relatively little effect on the observed dissociation. The use of equations derived for predicting the effects of dissociating reagents on the structure of subunit proteins [Herskovits, T. T., and Ibanez, V. S. (1976), Biochemistry 15, 5715] together with Setschenow constants based on the model amino acid data of Nandi and Robinson were found to give a satisfactory account of the dissociation behavior observed with many of the salts, giving reasonable estimates of the number of amino acids that form the smaller contact area of the alpha beta subunits of hemoglobin shown by the Perutz crystallographic model. The analysis of the dissociation data also extends the utility of the Setschenow constants tested for the characterization of the dissociation behavior of other subunit proteins.     

Lateral conductance parallel to membrane surfaces: effects of anesthetics and electrolytes at pre-transition.

The effects of dilute salts and anesthetics were studied on the impedance dispersion in the dipalmitoylphosphatidylcholine (DPPC) liposomes. Below the pre-transition temperature, the apparent activation energy for conductance in DPPC-H2O without salts was equivalent to pure water, 18.2 kJ mol-1. This suggests that the mobile ions (H3O+ and OH-) interact negligibly with the lipid surface below the pre-transition temperature. At pre-transition temperature, the apparent activation energy of the conductance decreased by the increase in the DPPC concentrations. The effects of various salts (LiCl, NaCl, KCl, KBr, and KI) on the apparent activation energy of the conductance were studied. Changes in anions, but not in cations, affected the activation energy. The order of the effect was Cl- less than Br- less than I-. Cations appear to be highly immobilized by hydrogen bonding to the phosphate moiety of DPPC. The smaller the ionic radius, the more ions are fixed on the surface at the expense of the free-moving species. The apparent activation energy of the transfer of ions at the vesicle surface was estimated from the temperature-dependence of the dielectric constant, and was 61.0 kJ mol-1 in the absence of electrolytes. In the presence of electrolytes, the order of the activation energy was F- greater than Cl- greater than Br- greater than I-. When the ionic radius is smaller, these anions interact with the hydration layer at the vesicle surface and the ionic transfer may become sluggish. In the absence of electrolytes, the apparent activation energy of the dielectric constant decreased by the increase in halothane concentrations. In the presence of electrolytes, however, the addition of halothane increased the apparent activation energy. We propose that the adsorption of halothane on the vesicle surface produces two effects: (1) destruction of the hydration shell, and (2) increase in the binding of electrolytes to the vesicle surface. In the absence of electrolytes, the first effect predominates and the apparent activation energy is decreased. In the presence of electrolytes, the latter effect predominates and the apparent activation energy is increased.     

The effect of chaotropic anions on the activation and the activity of spinach chloroplast fructose-1,6-bisphosphatase

The effect of chaotropic anions was studied on processes that constitute the chloroplast fructose-1,6-bisphosphatase reaction, i.e. enzyme activation and catalysis. The specific activity of chloroplast fructose-1,6-bisphosphatase was enhanced by preincubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotropic anion. When chaotropes were ranked in the order of increasing concentrations required for maximal activation they followed a lyotropic (Hofmeister) series: SCN- less than Cl3C-COO- less than ClO4- less than I- less than Br- less than Cl- less than SO4(2-). On the contrary, salts inhibited the catalytic step. The stimulation of chloroplast fructose-1,6-bisphosphatase by chaotropic anions arose from a decrease of the activation kinetic constants of both fructose 1,6-bisphosphate and Ca2+; on the other hand, in catalysis neutral salts caused a decrease of kcat because the S0.5 for both fructose 1,6-bisphosphate and Mg2+ remained unaltered. The molecular weight of chloroplast fructose-1,6-bisphosphatase did not change after the activation by incubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotrope; consequently, the action of these modulators altered the conformation of the enzyme. Modification in the relative position of aromatic residues of chloroplast fructose-1,6-bisphosphatase was detected by UV differential spectroscopy. In addition, the concerted action of modulators made the enzyme more sensitive to (a) trypsin attack and (b) S-carboxymethylation by iodoacetamide. These results provide a new insight on the mechanism of light-mediated regulation of chloroplast fructose-1,6-bisphosphatase; concurrently to the action of a sugar bisphosphate, a bivalent cation, and a reductant, modifications of hydrophobic interactions in the structure of chloroplast fructose-1,6-bisphosphatase play a crucial role in the enhancement of the specific activity.     

Interaction of rat lecithin-cholesterol acyltransferase with rat apolipoprotein A-I and with lecithin-cholesterol vesicles.

The interaction of rat plasma lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles and with rat apo-A-I was studied in comparison with that of human plasma lecithin-cholesterol acyltransferase to clarify the reaction mechanism of rat plasma lecithin-cholesterol acyltransferase. The interaction of both human and rat lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles was investigated by gel permeation chromatography on Superose 12. Both enzymes had almost the same affinity to the vesicles. The affinity of rat enzyme to rat apo-A-I was stronger than that of human enzyme to human apo-A-I when estimated on the apo-A-I-Sepharose 4B column. When human apo-A-I was added to the human enzyme/vesicle mixture which contained the enzyme-vesicle complex, the enzyme was effectively dissociated from the complex. But when rat apo-A-I was added to the rat enzyme/vesicle mixture, apo-A-I-enzyme-vesicle complex was still recognized by its elution pattern on gel permeation chromatography. This suggests that the mixture of rat enzyme, rat apo-A-I, and vesicles, which are the major components in the rat lecithin-cholesterol acyltransferase reaction, forms a stronger complex than do the components of the human reaction.     

Effects of extracellular anions on cell growth of Chinese hamster V-79 cells

The effects of extracellular anions (10-150 mM, added as Na salts to normal growth medium) on the growth of Chinese hamster V-79 cells were examined. Additions of NaCl and NaNO3 at concentrations greater than 60 mM reduced the growth rate dose-dependently. Several other anions also inhibited cell growth in the decreasing order of potency, SCN- greater than NO2- greater than NO3- greater than Br- greater than Cl- greater than gluconate- glutamate- greater than Mes-. When the added anions were removed, the growth rate was restored to the control rate. Cell survival was markedly reduced by the addition of SCN-, but was less affected by other anions (Cl-,NO3- and NO2-) of comparable potency. The respective syntheses of cellular DNA and protein, as estimated from the incorporation of [3H]-thymidine and [14C]leucine, also decreased with the increase in the concentration (60-120 mM) of anions added, the order of potency being SCN- greater than NO2- greater than NO3- greater than Cl-. After anion-treatment, the cellular Na+ concentration increased and the cellular Cl- concentration decreased in the order of SCN- greater than NO2- greater than NO3-, Cl-, but, the cellular K+ concentration did not change significantly. These data suggest that changes in extracellular anions affect cell growth and survival, probably through changes in the intracellular Na+ or Cl- concentration and in the rates of protein and/or DNA synthesis.     

Comparative study of tyrosinases from different sources: relationship between halide inhibition and the enzyme active site

The inhibition of tyrosinases from frog epidermis (Rana esculenta ridibunda), mushroom (Agaricus bisporus) and Harding-Passey mouse melanoma by halides is compared. In all cases, the inhibition is pH dependent, increasing when the pH decreases. The order of inhibition is I- greater than Br- greater than Cl- much greater than F- for frog epidermis tyrosinase, F- greater than I- greater than Cl- greater than Br- for mushroom tyrosinase and F- greater than Cl- much greater than Br- greater than I- for the mouse melanoma enzyme. These results are discussed in terms of the active site accessibility to exogenous ligands. The activation energies of the enzyme-catalysed L-dopa oxidation were also calculated, being the values 6.86, 17.01 and 20.25 kcal/mol for frog epidermis, mushroom and Harding-Passey mouse melanoma, respectively. A relationship between these values and the evolutionary adaptation of these enzymes is proposed.     

Chromophore equilibria in bacteriorhodopsin.

An investigation of the dark equilibria between different chromophores of bacteriorhodopsin (BR) and studies of the kinetics of their interconversion and photochemical activity have led to the following conclusions. (a) A component of the 605-nm chromophore of BR decays in the millisecond range and is likely to be identical to the intermediate O of the photochemical cycle of BR and is assumed to be formed from the purple complex (PC) by the binding of one proton to BR. (b) An acidic form the PC, PCaL-, arises from the 605-nm chromophore by selective binding of anions L- (F- greater than Cl- greater than Br- greater than I- greater than Cl04-) to BR. (c) The isomeric equilibrium between 13-cis and all-trans retinal is approximately 0.15/0.85 in PCaCl-, 0.3/0.7 in the 605-nm chromophore as compared to 0.5/0.5 in the PC. (d) The 500-nm chromophore is formed from the PC by release of nearly one proton from BR. (e) The pH range in which the PC exists is reduced in a high-temperature structure of the purple membrane as compared to its low temperature structure. A model for the chromophore structure is proposed as a hypothesis, which allows a comprehensive interpretation of the results. In this model the absorption spectrum of the retinylidene lysine Schiff base is modulated by its protonation state and the interaction with an anionic group.     

Influence of anions and cations on the dipole potential of phosphatidylcholine vesicles: a basis for the Hofmeister effect

Anions and cations have long been recognized to be capable of modifying the functioning of various membrane-related physiological processes. Here, a fluorescent ratio method using the styrylpyridinium dyes, RH421 and di-8-ANEPPS, was applied to determine the effect of a range of anions and cations on the intramembrane dipole potential of dimyristoylphosphatidylcholine vesicles. It was found that certain anions cause a decrease in the dipole potential. This could be explained by binding within the membrane, in support of a hypothesis originally put forward by A. L. Hodgkin and P. Horowicz [1960, J. Physiol. (Lond.) 153:404-412.] The effectiveness of the anions in reducing the dipole potential was found to be ClO4- > SCN- > I- > NO3- > Br- > Cl- > F- > SO42-. This order could be modeled by a partitioning of ions between the membrane and the aqueous phase, which is controlled predominantly by the Gibbs free energy of hydration. Cations were also found to be capable of reducing the dipole potential, although much less efficiently than can anions. The effects of the cations was found to be trivalent > divalent > monovalent. The cation effects were attributed to binding to a specific polar site on the surface of the membrane. The results presented provide a molecular basis for the interpretation of the Hofmeister effect of lyotropic anions on ion transport proteins.     

Nature of the enhancement of lecithin-cholesterol acyltransferase reaction by various apolipoproteins

The effects of human apolipoproteins on the lecithin-cholesterol acyltransferase reaction were studied by using purified human lecithin-cholesterol acyltransferase and phosphatidylcholine-cholesterol vesicles. When the assay mixtures contained an optimal amount or excess of apo-A-I, the addition of apo-A-II, apo-C-II, apo-C-III1, or apo-C-III2 inhibited the enzymatic reaction. However, at suboptimal apo-A-I concentrations, the addition of low concentrations of these apolipoproteins exhibited activating effects. The relative activating effects were greater at lower apo-A-I levels. Under no circumstance did the combined activating effect of apo-A-I and other apolipoproteins exceed the maximum activating effect observed with the optimal level of apo-A-I alone. Since apo-A-II, apo-C-II, and apo-C-III did not show significant activating effects in the absence of apo-A-I, these apolipoproteins apparently did not act as true activator proteins for the enzymatic reaction. The activation of the enzymatic reaction by apo-A-I alone was shown to be due in part to the enhancement of the enzyme transfer between the substrate particles. The replacement of the transfer-enhancing effect of apo-A-I by apo-A-II, apo-C-II, or apo-C-III appears to be responsible for their apparent activating effects in the presence of suboptimal levels of apo-A-I. These apolipoproteins seemed to coexist with both the enzyme and apo-A-I on the substrate particles under the conditions when they showed the activating effect. However, at the concentrations inhibitory to the enzymatic reaction, these apolipoproteins displaced both the enzyme and apo-A-I from the phosphatidylcholine-cholesterol vesicles.     

Anion-induced conformational change of apo-electron-transferring flavoprotein.

Apoprotein of electron-transferring flavoprotein (ETF) exists in an equilibrium between two different forms, only one of which can associate with FAD (Sato, K. et al. (1991) J. Biochem. 109, 734-740), as represented in the following kinetic scheme: A* in equilibrium with A, A+FAD in equilibrium with holoETF, where "A*" and "A" are the different forms of apoETF. In the present study, the effects of various anions on the conversion between the two forms of apoETF were investigated by kinetic analyses on binding of FAD to apoETF. All the anions tested here induced the conversion from "A*" to "A"; the order of the effectiveness was I- approximately Br- greater than Cl- greater than F-. Further, glycerol also induced the conversion from "A*" to "A". The elution pattern of apoETF on molecular sieve chromatography was changed by addition of salts or glycerol; this change was due to the conversion from "A*" to "A" by the added solutes. The "A*" form was eluted more rapidly than the "A" form, indicating that the "A*" form exists in a looser conformation than the "A" form. The far-UV CD spectral change upon addition of salts indicated that a greater part of the secondary structure is retained in the conversion from "A*" to "A," but the "A" form contains a somewhat larger amount of beta-sheet than "A*."     

Influence of protein conformation on its adaptability under chaotropic conditions

The thermostability of serum albumin and beta-lactoglobulin in various salt solutions was studied using differential scanning calorimetry. Below 1.0 M salt concentrations, the relative effectiveness of various sodium salts on increasing the thermostability of beta-lactoglobulin followed the classic Hofmeister or lyotropic series, i.e. SO2-(4) greater than Cl- greater than Br- greater than ClO-4 greater than SCN-; however, in the case of serum albumin the above order was reversed, i.e. ClO-4 greater than SCN- greater than Br- greater than Cl- greater than SO2-(4), indicating that the thermostability of serum albumin was higher in chaotropic solution conditions. Circular dichroic analysis of serum albumin in NaClO4 solutions revealed that the alpha-helical content of the protein increased from 59% to 73% in 1.0 M NaClO4; no similar increase in secondary structure was observed for beta-lactoglobulin. These observations contradicted the general notion that the chaotropic effect of neutral salts on the stability of macromolecules is independent of any details of the macromolecular conformation itself. The results presented here indicate that the predisposition of the native conformation of a protein per se might affect whether the protein would undergo stabilization or destabilization (i.e. conformational adaptability) under moderate chaotropic solution conditions.     

Competitive inhibition of liver glucokinase by its regulatory protein

The regulatory protein of rat liver glucokinase (hexokinase IV or D) behaved as a fully competitive inhibitor of this enzyme when glucose was the variable substrate, i.e. it increased the half-saturating concentration of glucose as a linear function of its concentration without affecting V (velocity at infinite concentration of substrate). The inhibition by the regulatory protein and that by palmitoyl-CoA were synergistic with that by N-acetyl-glucosamine, indicating that the two former inhibitors bind to a site distinct from the catalytic site. In contrast, the effects of the regulatory protein and palmitoyl-CoA were competitive with each other, indicating that these two inhibitors bind to the same site. The regulatory protein exerted a non-competitive inhibition with respect to Mg-ATP at concentrations of this nucleotide less than 0.5 mM. At higher concentrations, the latter antagonized the inhibition by the regulatory protein partly by decreasing the apparent affinity for fructose 6-phosphate. The following anions inhibited glucokinase non-competitively with respect to glucose: Pi, sulfate, I-, Br-, No3-, Cl-, F- and acetate. Pi and sulfate, at concentrations in the millimolar range, decreased the inhibition by the regulatory protein by competing with fructose 6-phosphate. Monovalent anions also antagonized the inhibition by the regulatory protein with the following order of potency: I- greater than Br- greater than NO3- greater than Cl- greater than F- greater than acetate and their effect was non-competitive with respect to fructose 6-phosphate. Glucokinase from Buffo marinus and pig liver were, like the rat liver enzyme, inhibited by the regulatory protein, as well as by palmitoyl-CoA at micromolar concentrations. In contrast, neither compound inhibited hexokinases from rat brain, beef heart or yeast, or the low-Km specific glucokinase from Bacillus stearothermophilus.     

Kinetic Analysis of the Chloride Dependence of the Neuronal Uptake of Dopamine and Effect of Anions on the Ability of Substrates to Compete with the Binding of the Dopamine Uptake Inhibitor GBR 12783

The specific binding of [3H]1-[2-(diphenyl-methoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([3H]GBR 12783) to the dopamine (DA) neuronal carrier present in membranes prepared from rat striatum was not affected when Cl- was replaced by either Br- or NO3-. In media containing Cl-, Br-, or NO3-, d-amphetamine and DA competed with the radioligand in a monophasic manner with Hill coefficients of close to 1 (0.94-1.12). Replacement of Cl- by Br- impaired the ability of some substrates (d-amphetamine, DA, p-hydroxyamphetamine, and m-tyramine) to compete with [3H]GBR 12783. The potency of Br- to decrease the affinity of substrates for the specific binding site was significantly correlated (t = 7.07, p less than 0.001) with their affinity for this binding site. These results suggest that the various substrates tested could bind to recognition sites in which Cl- is differently involved; as a consequence, substrates could bind to the neuronal carrier by means of partly different links. In experiments dealing with the specific uptake of [3H]DA, F-, NO3-, isethionate-, or acetate- was unable to substitute for Cl-, whereas Br- was quite a total substitute. Replacement of Cl- by equimolar concentrations of either NO3- or isethionate- resulted in inhibition curves of DA specific uptake with Hill coefficients of close to 1 (0.77 and 1.04 respectively); this indicates that both NO3- and isethionate- are devoid of inhibitory effects on neuronal uptake and are quite ineffective substitutes for Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)