Interfacial thermodynamics of protein adsorption, ion co-adsorption and ion binding in solution. II. Model interpretation of ion exchange in lysozyme chromatography

In this paper we present a model for the ion exchange effects in protein adsorption. The model is applied to chromatography of lysozyme on strong cation exchanger ‘mono S’. The experimental and general thermodynamic aspects have been discussed in Part 1, the preceding paper. The main modelling assumptions are (i) the charge regulation is confined to the small layer of contact between adsorbed protein and exchanger surface, (ii) the contact layer as a whole is electroneutral and (iii) the number of protein acid/base groups and exchanger surface acid groups which participate in the ion exchange is proportional to the area of the contact layer. The model is fitted to the experimental data by adjustment of only two or three parameters. The experimental co-adsorption numbers are very well reproduced. A few conspicuous features emerge: (i) the number of protein acid/base groups and exchanger surface acid groups in the contact layer varies with the medium conditions, such that the number is higher when the interaction between protein and exchanger surface is stronger. (ii) There is indirect evidence for structural alterations in the upper layers of the exchanger surface: the adsorbed protein is probably partly ‘buried’ in the surface.     

Connections: heart disease, cellular electrophysiology, and ion channels.

Our purpose in this article is to examine the hypothesis that both myocardial disease and ischemia can alter the electrophysiologic function of the ion channels responsible for the cellular electrical activity of the heart. Changes in the intracellular and extracellular milieus occur during ischemia and can alter the electrophysiology of several species of ionic channels and the cellular electrophysiologic activity of cardiac myocytes. Included are 1) changes in extracellular [K+] and pH and in intracellular [Na+], [Ca2+], and pH; 2) accumulation of noxious metabolic products such as lysophosphatidylcholine; and 3) depletion of intracellular ATP. Finally, ischemia or disease (e.g., hypertrophy) can alter the electrophysiology of at least two types of K+ channels, the A-like channels underlying the transient outward current and the inward rectifier, by mechanisms that apparently do not involve alteration of either the intra- or extracellular milieus. Findings suggest that the expression of cardiac A-like channel function can be altered by hypertrophy and that at least one intrinsic conductance property of the inward rectifier can be altered by ischemia. We speculate that the control of expression, function, and regulation of cardiac ion channels can be affected at the molecular level by heart disease and myocardial ischemia.     

Steady-state voltages, ion fluxes, and volume regulation in syncytial tissues.

Equations are developed that describe the steady-state relationships among ion fluxes, solute fluxes, water flow, voltage, concentration of solute, and hydrostatic pressure in a spherically symmetrical syncytial tissue. Each cell of the syncytium is assumed to have membrane channels for Na, K, and Cl, a membrane pump for Na/K, and some concentration of intracellular protein of net negative charge. However, the surface cells and inner cells of the tissue are assumed to have different distributions of membrane transport properties, hence there is a radial circulation of fluxes and a radial distribution of forces. Some reasonable approximations are made that allow analytic solutions of the nonlinear differential equations. These solutions are used to analyze data from the frog lens and are shown to account for the known steady-state properties of this tissue. Moreover, these solutions are used to make predictions on other steady-state properties, which have not been directly measured, and graphical results on the circulation of water, ions and solute through the frog lens are presented.     

Relationships between nitric oxide, nitroxyl ion, nitrosonium cation and peroxynitrite.

This review is concerned mainly with the three redox-related, but chemically distinct, species NO-, NO. and NO+, with greatest emphasis being placed on the chemistry and biology of the nitroxyl ion. Biochemical routes for the formation of nitroxyl ion and methods for showing the intermediacy of this species are discussed, together with chemical methods for generating nitroxyl ion in solution. Reactions of nitroxyl ion with NO., thiols, iron centres in haem and with dioxygen are reviewed The significance of the reaction between NO- and dioxygen as a source of peroxynitrite is assessed, and attention drawn to the possible significance of the spin state of the nitroxyl ion in this context. The biological significance of nitrosation and the importance of S-nitrosothiols and certain metal nitrosyl complexes as carriers of NO+ at physiological pH is stressed. Some features in the chemistry of peroxynitrite are noted.     

Gramicidins A, B, and C form structurally equivalent ion channels.

The membrane structure of the naturally occurring gramicidins A, B, and C was investigated using circular dichroism (CD) spectroscopy and single-channel recording techniques. All three gramicidins form channels with fairly similar properties (Bamberg, E., K. Noda, E. Gross, and P. L?uger. 1976. Biochim. Biophys. Acta. 419:223-228.). When incorporated into lysophosphatidylcholine micelles, however, the CD spectrum of gramicidin B is different from that of gramicidin A or C (cf. Prasad, K. U., T. L. Trapane, D. Busath, G. Szabo, and D. W. Urry. 1983. Int. J. Pept. Protein Res. 22:341-347.). The structural identity of the channels formed by gramicidin B has, therefore, been uncertain. We find that when gramicidins A and B are incorporated into dipalmitoylphosphatidylcholine vesicles, their CD spectra are fairly similar, suggesting that the two channel structures could be similar. In planar bilayers, gramicidins A, B, and C all form hybrid channels with each other. The properties of the hybrid channels are intermediate to those of the symmetric channels, and the appearance rates of the hybrid channels (relative to the symmetric channels) corresponds to what would be predicted if all three gramicidin molecules were to form structurally equivalent channels. These results allow us to interpret the different behavior of channels formed by the three gramicidins solely on the basis of the amino acid substitution at position 11.     

Interfacial thermodynamics of protein adsorption, ion co-adsorption and ion binding in solution. I. Phenomenological linkage relations for ion exchange in lysozyme chromatography and titration in solution

In this paper we discuss the thermodynamics of ion binding in solution, protein adsorption and ion co-adsorption. The emphasis is on charge regulation effects. To this end, we introduce phenomenological linkage relations from which the ion binding can be calculated from the electrolyte dependency of proton titration curves and the co-adsorption from the electrolyte dependency of protein adsorption isotherms. The linkage relations are derived from classical interfacial thermodynamics, and thus offer an alternative approach as compared to the mass balance equations which are currently used in biotechnology, and Record et al.'s 1978 analysis of Wyman's Binding Polynomial for protein interactions. The co-adsorption theory is an extension of our previous analysis of ion binding in solution, which we include here for comparison of the ion co-adsorption with the ion binding in solution. The theory is applied to the chromatography of lysozyme on the strong cation exchanger ‘mono S’ and to the proton titration of lysozyme in solution. In the accompanying Part 2 of this paper the results are interpreted with a simple model.     

Potential, pH, and arachidonate gate hydrogen ion currents in human neutrophils.

Indirect evidence indicates that a proton-selective conductance is activated during the respiratory burst in neutrophils. A voltage- and time-dependent H(+)-selective conductance, gH, in human neutrophils is demonstrated here directly by the whole-cell patch-clamp technique. The gH is extremely low at large negative potentials, increases slowly upon membrane depolarization, and does not inactivate. It is enhanced at high external pH or low internal pH and is inhibited by Cd2+ and Zn2+. Arachidonic acid, which plays a pivotal role in inflammatory reactions, amplifies the gH. The properties of the gH described here are compatible with its activation during the respiratory burst in stimulated neutrophils, in which it may facilitate sustained superoxide anion release by dissipating metabolically generated acid.     

Novel ion channels in the protists, Mougeotia and Saprolegnia, using sub-gigaseals.

Protoplasts of the filamentous alga, Mougeotia, and the filamentous fungal oomycete, Saprolegnia ferax, exhibit two K⁺ ion channels (2–6 pA) using the patch-clamp technique when the seals are less than 1 GΩ (about 100 MΩ). The membrane potential of the protoplasts was near 0 mV as measured intracellularly with double-barreled micropipettes; thus, inward K⁺ flux is due solely to concentration differences. Although conductances are in the range expected for K⁺ channels, the activity at 0 mV is not seen in other organisms under gigaseal conditions. This paper draws attention to the usefulness of this subsidiary patch-clamp technique and the novel characteristics of ion channels in Mougeotia and Saprolegnia.     

ADP, chloride ion, and metal ion binding to bovine brain glutamine synthetase

The binding of divalent cations and nucleotide to bovine brain glutamine synthetase and their effects on the activity of the enzyme were investigated. In ADP-supported gamma-glutamyl transfer at pH 7.2, kinetic analyses of saturation functions gave [S]0.5 values of approximately 1 microM for Mn2+, approximately 2 mM for Mg2+, 19 nM for ADP.Mn, and 7.2 microM for ADP.Mg. The method of continuous variation applied to the Mn2+-supported reaction indicated that all subunits of the purified enzyme express activity when 1.0 equiv of ADP is bound per subunit. Measurements of equilibrium binding of Mn2+ to the enzyme in the absence and presence of ADP were consistent with each subunit binding free Mn2+ (KA approximately equal to 1.5 X 10(5) M-1) before binding the Mn.ADP complex (KA' approximately equal to 1.1 X 10(6) M-1). The binding of the first Mn2+ or Mg2+ to each subunit produces structural perturbations in the octameric enzyme, as evidenced by UV spectral and tryptophanyl residue fluorescence changes. The enzyme, therefore, has one structural site per subunit for Mn2+ or Mg2+ and a second site per subunit for the metal ion-nucleotide complex, both of which must be filled for activity expression. Chloride binding (KA' approximately equal to 10(4) M-1) to the enzyme was found to have a specific effect on the protein conformation, producing a substantial (30%) quench of tryptophanyl fluorescence and increasing the affinity of the enzyme 2-4-fold for Mg2+ or Mn2+. Arsenate, which activates the gamma-glutamyl transfer activity by binding to an allosteric site, and L-glutamate also cause conformational changes similar to those produced by Cl- binding. Anion binding to allosteric sites and divalent metal ion binding at active sites both produce tryptophanyl residue exposure and tyrosyl residue burial without changing the quaternary enzyme structure.     

Metal ion transporters and homeostasis.

Transition metals are essential for many metabolic processes and their homeostasis is crucial for life. Aberrations in the cellular metal ion concentrations may lead to cell death and severe diseases. Metal ion transporters play a major role in maintaining the correct concentrations of the various metal ions in the different cellular compartments. Recent studies of yeast mutants revealed key elements in metal ion homeostasis, including novel transport systems. Several of the proteins discovered in yeast are highly conserved, and defects in some of the yeast mutants could be complemented by their human homologs. The studies of yeast metal ion transporters helped to unravel the molecular mechanism of macrophage defense against bacterial infection and hereditary diseases.     

Metabolic modulation of neurotransmitter release--adenosine, adenine nucleotides, potassium, hyperosmolarity, and hydrogen ion.

Evidence has accumulated that several factors, which have been proposed as mediators of exercise hyperemia, can modulate adrenergic neurotransmission in blood vessels. Adenosine and the adenine nucleotides depress the response of isolated blood vessels of the dog to nerve stimulation more than that to exogenous norepinephrine; this difference is explained by a decreased release of the neurotransmitter. Potassium, hyperosmolarity, and acidosis also depress adrenergic neurotransmission in isolated veins. These results are consistent with the hypothesis that metabolic changes in the vicinity of the adrenergic neuroeffector junction are capable of decreasing the output of neurotransmitter to the blood vessels in the exercising muscle.     

Salinity tolerance of Kosteletzkya virginica. I. Shoot growth, ion and water relations

Abstract. Kosteletzkya virginica (L.) Presl., a dicotyledonous halophyte native to brackish tidal marshes, was grown on nutrient solution containing 0. 85, 170 or 255 mol m^-3 NaCl, and the effects of external salinity on shoot growth and ion content of individual leaves were studied in successive harvests. Growth was stimulated by 85 mol m^-3 NaCl and was progressively reduced at the two higher salinities. Growth suppression at high salinity resulted principally from decreased leaf production and area, not from accelerated leaf death. As is characteristic of halophytic dicots. K. virginica accumulated inorganic ions in its leaves, particularly Na⁺ and K⁺. However, the Na⁺ concentration of individual leaves did not increase with time, but remained constant or even declined, seeming to be well-coordinated with changes in water content. A striking feature of the ion composition of salinized plants was the development of a dramatic gradient in sodium content, with Na⁺ partitioned away from the most actively growing leaves. Salt-treated plants exhibited a strong potassium affinity, with foliar K⁺ levels higher in salinized plants than unsalinized plants after an initial decrease. These results suggest that selective uptake and transport, foliar compartmentation of Na⁺ and K⁺ in opposite directions along the shoot axis, and the regulation of leaf salt loads over time to prevent build-up of toxic concentrations are whole-plant features which enable K. virginica to establish favourable K⁺-Na⁺ relations under saline conditions.     

Salinity tolerance of Kosteletzkya virginica. II. Root growth, lipid content, ion and water relations

Abstract. Kosteletzkya virginica (L.) Presl., a dicot halophyte native to brackish tidal marshes, was grown on nutrient solution containing 0. 85, 170 or 255 mol m ³ NaCl, and the effects of external salinity on root growth, ion and water levels, and lipid content were examined in successive harvests. Root growth paralleled shoot growth trends, with some enhancement observed at 85 mol m ³ NaCl and a reduction noted at the higher salinities. Root Na⁺ content increased with increasing external NaCl, but remained constant with time for each treatment. K⁺ content, although lower in salt-grown plants after 14 d salinization, subsequently increased to levels comparable to unsalinized plants. A strong K⁺ affinity was reflected in the increased K⁺/Na⁺ selectivity of salt-grown plants and by their low Na⁺/K⁺ ratios. Cl levels rose in salinized plants and values were double or more those for Na⁺, indicating the possibility of a sodium-excluding mechanism in roots. Root phospholipids and sterols, principal membrane constituents, were maintained or elevated and the free sterol/phospholipids ratio increased in salinized K. virginica plants, suggesting retention of overall membrane structure and decreased permeability. This response, considered in light of root calcium maintenance and high potassium levels, suggests that salinity-induced changes in membrane lipid composition may be important in preventing K⁺ leakage from cells.     

Interaction of cytochrome c, ferrous ion, and phosphate. Electron transfer within a stoichiometric complex.

The rate and extent of electron transfer from ferrous ion to ferricytochrome c are enhanced by the presence of inorganic orthophosphate at concentrations comparable to those of reductant and oxidant. Evidence, obtained by the method of continuous variations, shows that the electron transfer occurs within a stoichiometric complex composed of cytochrome c, ferrous ion, and phosphate in molar proportions of about 1:1:1. The incorporation of the anion into this complex appears to result in a modulation of the extent and rate of cytochrome c reduction. The rate of electron transfer obeys a first order rate law, characterized by an apparent first order rate constant of 1.4 min-1. The complex has kinetic significance only; equilibrium dialysis, gel filtration, and sedimentation velocity experiments yielded no evidence for stable binding of phosphate and iron, or of aggregation, on a significant scale. The extent of reduction is limited (for reasons not yet known) to about one-half of the available cytochrome molecules. Reduction in excess of 50% can be achieved only when both, ferrous ion and phosphate, are present in excess of the cytochrome concentration. Kinetic data indicate that reduction to extents over and below 50% occurs by different mechanisms.