Nuclear-magnetic-resonance studies of carboxypeptidase B. Binding of inhibitors to the manganese enzyme.

Longitudinal and transverse proton relaxation rates of water in solutions of porcine manganese carboxypeptidase B have been measured in the presence of various competitive inhibitors by pulse nuclear magnetic resonance (NMR) spectrometry. The inhibition constant of Mn-carboxypeptidase activity by L-argininic acid and acetyl-L-arginine was in agreement with the equilibrium constant obtained by the NMR method, indicating similar and specific binding of the inhibitors to the active site of the manganese enzyme. Titration of the water boound to the metal ion revealed the presence of one water molecular which could be displaced from the sphere of the managenese ion by various inhibitors. The structural features of the inhibitors required for this displacement as well as the mode of interaction is described.     

Preservation of the diffusible cations for secondary ion mass spectrometry. II. Artefacts in material embedded in araldite or melamine.

Flotation on hot water (about 60 degrees C) which is frequently employed to stretch semithin sections on substrates for SIMS (secondary ion mass spectrometry) microscopy, is the cause of numerous artefacts. In the case of epoxy resin-embedded tissue, one observes loss of potassium and sodium and accumulation of calcium. The relative contrast of cell nuclei in the ionic images, is rapidly affected by these ion migrations. After prolonged contact with hot water, tissue becomes uniformly emissive. In the case of hydrosoluble resin-embedded tissue, potassium and sodium do not appear to be affected by the action of water, which suggests that they are covalently bound with chelating sites buried beneath the layer of water bound to the surface of the macromolecules. Calcium accumulates, probably on widely exposed anionic sites. Moreover, the domains observed in hydrosoluble resin-embedded tissue shrink differently according to the proportion of water removed by melamine; this can provide interesting information on the initial equilibrium between water, ion sand macromolecules. Our results seem to support the assumption that bound water should play an important role in the preservation of both macromolecular architecture and ion distributions.     

Ion coordination in the amphotericin B channel.

The antifungal polyene antibiotic amphotericin B forms channels in lipid membranes that are permeable to ions, water, and nonelectrolytes. Anion, cation, and ion pair coordination in the water-filled pore of the "barrel" unit of the channels was studied by molecular dynamics simulations. Unlike the case of the gramicidin A channel, the water molecules do not create a single-file configuration in the pore, and some cross sections of the channel contain three or four water molecules. Both the anion and cation are strongly bound to ligand groups and water molecules located in the channel. The coordination number of the ions is about six. The chloride has two binding sites in the pore. The binding with water is dominant; more than four water molecules are localized in the anion coordination sphere. Three motifs of the ion coordination were monitored. The dominant motif occurs when the anion is bound to one ligand group. The ion is bound to two or three ligand groups in the less favorable configurations. The strong affinity of cations to the channel is determined by the negatively charged ligand oxygens, whose electrostatic field dominates over the field of the hydrogens. The ligand contribution to the coordination number of the sodium ion is noticeably higher than in the case of the anion. As in the case of the anion, there are three motifs of the cation coordination. The favorable one occurs when the cation is bound to two ligand oxygens. In the less favorable cases, the cation is bound to three or four oxygens. In the contact ion pair, the cation and anion are bound to two ligand oxygens and one ligand hydrogen, respectively. There exist intermediate solvent-shared states of the ion pair. The average distances between ions in these states are twice as large as that of the contact ion pair. The stability of the solvent-shared state is defined by the water molecule oriented along the electrostatic field of both ions.     

Molecular dynamics simulation of cation motion in water-filled gramicidinlike pores.

A model calculation is carried out to study the potential energy profile of a sodium ion with several water molecules inside a simplified model of the gramicidin ion channel. The sodium ion is treated as a Lennard-Jones sphere with a point charge at its center. The Barnes polarizable water model is used to mimic the water molecules. A polarizable and deformable gramicidinlike channel is constructed based on the model obtained by Koeppe and Kimura. Potential minima and saddle points are located and the static energy barriers are computed. The potential minima at the two mouths of the channel exhibit an aqueous solvation structure very different from that at any of the interior minima. These sites are approximately 23.6 and 24.4 A apart for binding of a sodium ion and a cesium ion, respectively. Ionic motion from these exterior sites to the first interior minimum requires substantial rearrangement of the waters of solvation; this rearrangement may be the hydration/dehydration step in ionic permeation through the channel. Based on these results, a mechanism by which the sodium ion moves from the exterior binding site to the interior of the channel is proposed. Our model channel accommodates about eight water molecules and the transport of the ion and water within the channel is found to be single file. Results of less extensive calculations for Cs+ and Li+ ions in a channel with or without water are also reported.     

Ion dependence of the tarsal sugar receptor of the blowfly Phormia regina.

The activity of the tarsal sugar receptor is greatly reduced following prolonged water exposure. The animal's behavior, which characteristícally reflects receptor input, also shows decreased acceptance of sucrose solutions following prolonged tarsal immersion in deionized water. Long exposure of the tarsi to Bodenstein's saline instead of water does not produce as large a decrement in the acceptance response as does water exposure.Recovery of the behavioral response occurs spontaneously after a few hours. The original response level can also be restored immediately if a moderate concentration (0.05 to 0.2 M) of KCl or NaCl is added to the sucrose stimulus. The effect of LiCl is ambiguous: it inhibits the normal sucrose response, thereby tending to mask any restorative effects. The electrophysiological data show that the cellular response level is also restored when Na⁺ or K⁺ ions are present in the stimulus.The above data are interpreted to mean that the effect of tarsal water exposure is to slowly leach out ions in the effective extracellular fluid surrounding the receptor membrane, thus lowering the membrane potential and deceasing the receptor potential upon stimulation. The fact that Na⁺ and K⁺ when supplied in the stimulating solution temporarily restore the original response level suggests that these extrinsically added ions can be used as current carrying ions to depolarize the cell. The data suggest that the sensillum contains three functional compartments interconnected by partial diffusion barriers: (1) a ‘receptor compartment’ (2) an axial cylinder which contains the dendrites and functions as the immediate extracellular ion source, and (3) a larger axial cylinder which serves as an ion reservoir.A method for statistically analyzing behavioral acceptance data is presented.     

Energetics of osmoregulation: II. Water flux and osmoregulatory work in the euryhaline fish, Fundulus heteroclitus

Teleost fish experience passive osmotic water influx in fresh water (FW) and water outflux in salt water, which is normally compensated by water flow driven by active ion transport mechanisms. Euryhaline fish may also minimize osmotic energy demand by "behavioral osmoregulation", seeking a medium isotonic with their body fluids. Our goal was to evaluate the energy requirement for osmoregulation by the euryhaline fish Fundulus heteroclitus, to determine whether it is of sufficient magnitude to favor behavioral osmoregulation. We have developed a method of weighing small fish repetitively for long periods without apparent damage, which was used to assess changes in water content following changes in external salinity. We found that cold (4 degrees C) inhibits osmoregulatory active transport mechanisms in fish acclimated to warmer temperatures, leading to a net passive water flux which is reversed by rewarming the fish. A sudden change of salinity at room temperature triggers a transient change in water content and the initial slope can be used to measure the minimum passive flux at that temperature. With some reasonable assumptions as to the stoichiometry of the ion transport and ATP-generating processes, we can calculate the amount of respiration required for ion transport and compare it to the oxygen uptake measured previously under the same conditions. We conclude that osmoregulation in sea water requires from 6% to 10% of the total energy budget in sea water, with smaller percentages in FW, and that this fraction is probably sufficient to be a significant selective driving force favoring behavioral osmoregulation under some circumstances.     

Sodium in gramicidin: an example of a permion.

The reaction path and free energy profile of Na+ were computed in the interior of the channel protein gramicidin, with the program MOIL. Gramicidin was represented in atomic detail, but surrounding water and lipid molecules were not included. Thus, only short range interactions were investigated. The permeation path of the ion was an irregular spiral, far from a straight line. Permeation cannot be described by motions of a single Na+ ion. The minimal energy path includes significant motion of water and channel atoms as well as motion of the permeating ion. We think of permeation as motion of a permion, a quasi-particle that includes the many body character of the permeation process, comparable with quasi-particles like holes, phonons, and electrons of solid-state physics. Na+ is accompanied by a plug of water molecules, and motions of water, Na+, and the atoms of gramicidin are highly correlated. The permion moves like a linear polymer made of waters and ion linked and moving coherently along a zigzag line, following the reptation mechanism of polymer transport. The effective mass, free energy, and memory kernel (of the integral describing time-dependent friction) of short range interactions were calculated. The effective mass of the permion (properly normalized) is much less than Na+. Friction varies substantially along the path. The free energy profile has two deep minima and several maxima. In certain regions, the dominant motions along the reaction path are those of the channel protein, not the permeating ion: there, ion waits while the other atoms move. At these waiting sites, the permion's motion along the reaction path is a displacement of the atoms of gramicidin that prepare the way for the Na+ ion.     

Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.     

Combined transport of water and ions through membrane channels

The coupling of ion and water flow through membrane channels is under dispute. Among all human aquaporins only aquaporin-6 exhibits ion channel activity. Whether aquaporin-6 functions also as a water channel cannot yet be determined with confidence. Similarly, a comparison of single-channel water permeabilities from ion channels and aquaporins suggests that ion channels may play a secondary role as water channels. However, the fraction of absorbed fluid that crosses epithelial ion channels still remains to be determined.     

Photosynthetic water oxidation. A new chemical model

A sequential four-step chemical model for the water oxidation process in photosystem II is presented, based on the observation that a peroxide-linked biquinone complex can be chemically formed as a result of hydroxide ion addition to quinone. In our model, the hydroxide ion intermediate is generated in photosystem II as a result of proton abstraction from water. In the model, the first two flashes of light raise the oxidation state of the bimanganese center, while the third and fourth flashes of light sequentially generate the peroxide-linked biquinone which is then directly oxidized by the bimanganese center to produce oxygen and regenerate quinone.     

Charge density-dependent strength of hydration and biological structure.

Small ions of high charge density (kosmotropes) bind water molecules strongly, whereas large monovalent ions of low charge density (chaotropes) bind water molecules weakly relative to the strength of water-water interactions in bulk solution. The standard heat of solution of a crystalline alkali halide is shown here to be negative (exothermic) only when one ion is a kosmotrope and the ion of opposite charge is a chaotrope; this standard heat of solution is known to become proportionally more positive as the difference between the absolute heats of hydration of the corresponding gaseous anion and cation decreases. This suggests that inner sphere ion pairs are preferentially formed between oppositely charged ions with matching absolute enthalpies of hydration, and that biological organization arises from the noncovalent association of moieties with matching absolute free energies of solution, except where free energy is expended to keep them apart. The major intracellular anions (phosphates and carboxylates) are kosmotropes, whereas the major intracellular monovalent cations (K+; arg, his, and lys side chains) are chaotropes; together they form highly soluble, solvent-separated ion pairs that keep the contents of the cell in solution.     

Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: a pentacoordinated binding of the SCN- ion to the zinc at high pH

The binding of four inhibitors--mercuric ion, 3-acetoxymercuri-4-aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion--to human carbonic anhydrase II (HCA II) has been studied with X-ray crystallography. The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 A resolution. Mercuric ions are observed at both nitrogens in the His-64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys-206. The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 A, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closest to the zinc ion is 3.1 A away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc. The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 A resolution using the least-squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN- ion is 1.9 A from the zinc ion but shifted 1.3 A with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the O gamma l atom of Thr-199. This is due to the inability of the O gamma l atom of Thr-199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN- molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so-called "deep" water molecule of the native enzyme. The zinc-bound water molecule is 2.2 A from the zinc ion and 2.4 A from the SCN- nitrogen. In addition, this water is hydrogen bonded to the O gamma l atom of Thr-199 and to another water molecule. We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3- ion are ligated to the zinc ion.     

Structure of cobalt carbonic anhydrase complexed with bicarbonate.

The three-dimensional structure of a complex between catalytically active cobalt(II) substituted human carbonic anhydrase II and its substrate bicarbonate was determined by X-ray crystallography (1.9 A). One water molecule and two bicarbonate oxygen atoms are found at distances between 2.3 and 2.5 A from the cobalt ion in addition to the three histidyl ligands contributed by the peptide chain. The tetrahedral geometry around the metal ion in the native enzyme with a single water molecule 2.0 A from the metal is therefore lost. The geometry is difficult to classify but might best be described as distorted octahedral. The structure is suggested to represent a water-bicarbonate exchange state relevant also for native carbonic anhydrase, where the two unprotonized oxygen atoms of the substrate are bound in a carboxylate binding site and the hydroxyl group is free to move closer to the metal thereby replacing the metal-bound water molecule. A reaction mechanism based on crystallographically determined enzyme-ligand complexes is represented.     

The molecular basis of water transport in the brain

Brain function is inextricably coupled to water homeostasis. The fact that most of the volume between neurons is occupied by glial cells, leaving only a narrow extracellular space, represents an important challenge, as even small extracellular volume changes will affect ion concentrations and therefore neuronal excitability. Further, the ionic transmembrane shifts that are required to maintain ion homeostasis during neuronal activity must be accompanied by water. It follows that the mechanisms for water transport across plasma membranes must have a central part in brain physiology. These mechanisms are also likely to be of pathophysiological importance in brain oedema, which represents a net accumulation of water in brain tissue. Recent studies have shed light on the molecular basis for brain water transport and have identified a class of specialized water channels in the brain that might be crucial to the physiological and pathophysiological handling of water.     

Water and polypeptide conformations in the gramicidin channel. A molecular dynamics study.

Theoretical studies of ion channels address several important questions. The mechanism of ion transport, the role of water structure, the fluctuations of the protein channel itself, and the influence of structural changes are accessible from these studies. In this paper, we have carried out a 70-ps molecular dynamics simulation on a model structure of gramicidin A with channel waters. The backbone of the protein has been analyzed with respect to the orientation of the carbonyl and the amide groups. The results are in conformity with the experimental NMR data. The structure of water and the hydrogen bonding network are also investigated. It is found that the water molecules inside the channel act as a collective chain; whereas the conformation in which all the waters are oriented with the dipoles pointing along the axis of the channel is a preferred one, others are also accessed during the dynamics simulation. A collective coordinate involving the channel waters and some of the hydrogen bonding peptide partners is required to describe the transition of waters from one configuration to the other.     

Energetics of ribonuclease A catalysis. 2. Nonenzymatic hydrolysis of cytidine cyclic 2',3'-phosphate

Various kinetic aspects of the nonenzymatic hydrolysis of cytidine cyclic 2',3'-phosphate and uridine cyclic 2',3'-phosphate have been studied in order to provide a basis for comparison with the ribonuclease A catalyzed hydrolysis reaction. Studies of the pH dependence of the nonenzymatic reaction reveal mechanisms that are first order in hydroxide concentration and second order in hydrogen ion concentration, in addition to a "water" reaction. The rate constant for the water reaction was found to be very small, approximately equal to 2.5 X 10(-6) min-1. General base catalyzed hydrolysis reactions were also studied with imidazole as the catalyst. At pH values in which both the protonated and neutral forms of imidazole are present, a kinetic mechanism was observed that appears to be second order in total imidazole concentration, thus suggesting that bifunctional catalysis occurs. The activation enthalpy for the hydroxide, hydrogen ion, water, and imidazole catalyzed reactions was determined.     

Plasmalemma-mitochondrial complexes involved in water transport in the hindgut of a milliped,Scaphiostreptus sp.

Summary Plasmalemma-mitochondrial complexes (PMC) are known to be very active in water transport in different tissues. The PMC in the hindgut cells of the milliped, Scaphiostreptus sp., differ from those found in other transporting epithelia of hindguts; they are closely connected with cisternae of the endoplasmic reticulum. This arrangement is thought to be very efficient in ion and water transport.     

Heavy water inhibition of the transport of alkaline cations across the muscle membrane. I. A comparison of different types of sodium transfer

The sodium efflux from the frog sartorius muscle into the media of different ion composition, prepared with ordinary and heavy water, was measured by radiotracer and flame-emission techniques. About the half of the sodium in muscles was substituted for lithium. The ouabain-sensitive, as well as external potassium- and external sodium-dependent components of the efflux were found to be totally inhibited in D2O, whereas the residual efflux observed in sodium- and potassium-free magnesium medium was diminished in D2O only by one half. A conclusion is made that the decrease in sodium efflux in D2O is due to the inhibition of sodium transfer through the Na, K-ATPase transport system.     

ON THE CAUSE OF THE INFLUENCE OF IONS ON THE RATE OF DIFFUSION OF WATER THROUGH COLLODION MEMBRANES. II

1. It had been shown in previous publications that when pure water is separated from a solution of an electrolyte by a collodion membrane the ion with the same sign of charge as the membrane increases and the ion with the opposite sign of charge as the membrane diminishes the rate of diffusion of water into the solution; but that the relative influence of the oppositely charged ions upon the rate of diffusion of water through the membrane is not the same for different concentrations. Beginning with the lowest concentrations of electrolytes the attractive influence of that ion which has the same sign of charge as the collodion membrane upon the oppositely charged water increases more rapidly with increasing concentration of the electrolyte than the repelling effect of the ion possessing the opposite sign of charge as the membrane. When the concentration exceeds a certain critical value the repelling influence of the latter ion upon the water increases more rapidly with a further increase in the concentration of the electrolyte than the attractive influence of the ion having the same sign of charge as the membrane. 2. It is shown in this paper that the influence of the concentration of electrolytes on the rate of transport of water through collodion membranes in electrical endosmose is similar to that in the case of free osmosis. 3. On the basis of the Helmholtz theory of electrical double layers this seems to indicate that the influence of an electrolyte on the rate of diffusion of water through a collodion membrane in the case of free osmosis is due to the fact that the ion possessing the same sign of charge as the membrane increases the density of charge of the latter while the ion with the opposite sign diminishes the density of charge of the membrane. The relative influence of the oppositely charged ions on the density of charge of the membrane is not the same in all concentrations. The influence of the ion with the same sign of charge increases in the lowest concentrations more rapidly with increasing concentration than the influence of the ion with the opposite sign of charge, while for somewhat higher concentrations the reverse is true.