Correlation between monovalent cation-induced decreases in chlorophyll a fluorescence and chloroplast structural changes

Low concentrations (~ 3 mm) of salts of monovalent cations such as Na⁺, K⁺, and tetraethylammonium were found to decrease the turbidity of chloroplast suspensions. The turbidity changes (Δ₅₄₀) had the same kinetics, salt concentration dependence, and pH dependence as the monovalent cation-induced decreases in chlorophyll a fluorescence (9), suggesting that structural changes are the cause of the associated increases in spillover. Electron microscopy revealed that the grana are stacked when spillover is inhibited (in the absence of salts or the presence of divalent cations) and that monovalent cations cause the grana to unstack, thereby promoting spillover.     

Assay and properties of the hemolysis activity of pure venom from the nematocysts of the acontia of the sea anemone Aiptasia pallida

Pure venom from the acontial nematocysts of the sea anemone Aiptasia pallida was isolated and an assay for the hemolysis activity of the venom devised. The assay is rapid, sensitive, and reproducible. Venom concentrations as low as 0.1 μg protein/ ml were accurately assayed. The properties of the hemolysis activity were analyzed using techniques similar to those used to study enzyme-catalyzed reactions. The biochemical events underlying venom-induced lysis required the direct participation of millimolar levels of Ca²⁺. The slight variability of the apparent K_m for Ca²⁺ at different venom concentrations appeared to be due to the release of some material(s) from lysing cells. Both Sr²⁺ and Mg²⁺ weakly substituted for Ca²⁺. Inhibition of lysis by EDTA was reversed by Ca²⁺. Small monovalent cations, such as Na⁺ or K⁺, appeared to be involved in the venom-induced alteration of the red cell membrane so that lysis could occur. The venom's hemolysis activity was stabilized in solution only if the concentration of the venom proteins was high while also in the presence of at least the equivalent of 0.15 m NaCl.     

Effects of monovalent cations on Ca uptake by skeletal and cardiac muscle sarcoplasmic reticulum

Ca²⁺ transport by the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is sensitive to monovalent cations. Possible K⁺ binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca²⁺ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca²⁺ transport correlated in most cases with their direct effects on SERCA. Choline⁺, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca²⁺ transport. Of the monovalent cations tested, only Cs⁺ significantly affected the Hill coefficient of Ca²⁺ transport (n_H). An increase in n_H from ∼2 in K⁺ to ∼3 in Cs⁺ was seen in all of the forms of SERCA examined. The effects of Cs⁺ on the maximum velocity of Ca²⁺ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K⁺ binding sites of the different forms of the protein.     

The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

Active-Site Monovalent Cations Revealed in a 1.55-Å-Resolution Hammerhead Ribozyme Structure

We have obtained a 1.55-Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under conditions that permit detailed observations of Na⁺ ion binding in the ribozyme's active site. At least two such Na⁺ ions are observed. The first Na⁺ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical with that previously observed for divalent cations. A second Na⁺ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with that of previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na⁺, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na⁺ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active-site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest-resolution ribozyme structure in the Protein Data Bank.     

Structural and Functional Model for Ionic (K/Na) and pH Dependence of GTPase Activity and Polymerization of FtsZ, the Prokaryotic Ortholog of Tubulin

Bacterial cell division occurs through the formation of a protein ring (division ring) at the site of division, with FtsZ being its main component in most bacteria. FtsZ is the prokaryotic ortholog of eukaryotic tubulin; it shares GTPase activity properties and the ability to polymerize in vitro. To study the mechanism of action of FtsZ, we used molecular dynamics simulations of the behavior of the FtsZ dimer in the presence of GTP-Mg²⁺ and monovalent cations. The presence of a K⁺ ion at the GTP binding site allows the positioning of one water molecule that interacts with catalytic residues Asp235 and Asp238, which are also involved in the coordination sphere of K⁺. This arrangement might favor dimer stability and GTP hydrolysis. Contrary to this, Na⁺ destabilizes the dimer and does not allow the positioning of the catalytic water molecule. Protonation of the GTP gamma-phosphate, simulating low pH, excludes both monovalent cations and the catalytic water molecule from the GTP binding site and stabilizes the dimer. These molecular dynamics predictions were contrasted experimentally by analyzing the GTPase and polymerization activities of purified Methanococcus jannaschii and Escherichia coli FtsZ proteins in vitro.     

On the mechanism of anion uptake by plant roots

Summary The effect of the valence of the associated cation on Cl^–-uptake by excised barley roots grown in CaSO₄ has been studied at 26°, 6° and 2°C. The uptake of Cl^– relative to that of the associated cation was found to increase in the order: trivalent > divalent > monovalent. This was explained on the expected effect of the cation on the negative charge and potential of root surfaces. A lyotropic order was observed in case of monovalent cations, whereas divalent cations showed no such order. The order observed in Cl^–-uptake from chloride solutions of monovalent cations is associated with the ability of the absorbed cation to remove Ca and Mg from the roots. Li⁺ behaved similar to divalent cations in affecting the relative Cl^–-uptake from LiCl.As to the effect of temperature on the uptake of Cl^– and associated cation, it appears that Cl^– is not taken up to any great extent at 2°C whereas cations are still adsorbed at this low temperature. This has been explained on the assumption of the presence of negative adsorption spots on the root surface which can hold cations but not anions. It appears that Cl^–-uptake by roots requires the expenditure of energy to overcome repulsion arising from the negative surface.This work is supported by AEC contract AT (11-1) — 34 project 55.     

Effect of Monovalent Cations on the Aggregation of Tobacco Mosaic Virus by Chondroitin Sulfate

The mechanism of precipitation of tobacco mosaic virus by chondroitin sulfate in the presence of various monovalent cations was investigated kinetically by means of turbidimetry. The virus solution became turbid on the addition of chondroitin sulfate, and led to separation of the virus as a crystalline phase. In the presence of monovalent cations the degree of precipitation of the virus by chondroitin was reduced with the increase in monovalent cation concentration. The order of the reduction was Li⁺<Na⁺<K⁺, which is reversely analogous to the lyotropic series. The least hydrated cation, K⁺, reduced the degree of precipitation of tobacco mosaic virus the most because the radius of the sphere equivalent to chondroitin sulfate was diminished most strongly by K⁺ ion. The effect of the monovalent cations on the precipitation supports our assumption that the virus precipitation results from incompatibility and mutual spatial exclusion between the virus and chondroitin. The maximum turbidities, the initial slopes, and the aggregation half-time were measured by varying the monovalent cation concentrations. The former two parameters decreased with the increase in cation concentration, whereas the aggregation half-time increased. A lag time was present and the effect of the cations increased in the order K⁺<Na⁺<Li⁺.     

Effects of Physicochemical Factors on the Adhesion to Cellulose Avicel of the Ruminal Bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes subsp. succinogenes

Ruminococcus flavefaciens adhered instantly to cellulose, while Fibrobacter succinogenes had the highest percentage of adherent cells after about 25 min of contact between bacteria and cellulose. Adhesion of R. flavefaciens was unaffected by high concentrations of sugars (5%), temperature, pH, oxygen, metabolic inhibitors, and lack of Na⁺. In contrast, the attachment was affected by the removal of divalent cations (Mg²⁺ and Ca²⁺), the presence of cellulose derivatives (methylcellulose and hydroxyethylcellulose), and cystine. Adhesion of F. succinogenes was sensitive to low and high temperatures, high concentrations of glucose and cellobiose (5%), hydroxyethylcellulose (0.1%), redox potential, pH, lack of monovalent cations, and the presence of an inhibitor of membrane ATPases or lasalocid and monensin. Cells of F. succinogenes heated at 100°C no longer were adherent. On the other hand, adhesion was insensitive to the lack of divalent cations (Mg²⁺ and Ca²⁺), the presence of 2,4-dinitrophenol, tetrachlorosalicylanilide, or inhibitors of the electron transfer chains. Adhesion of F. succinogenes seems to be related to the metabolic functions of the cell. External proteins and/or cellulases themselves might play a part in the attachment process. Several mechanisms are probably involved in the adhesion of R. flavefaciens, the main one being the interaction between the large glycocalyx and the divalent cations Ca²⁺ and Mg²⁺. Hydrophobic bonds and enzymes may also be involved.     

The calculation of partial specific volumes of proteins in guanidine hydrochloride

The effects of various modifiers on the ATPase activity of bovine platelet actomyosin has been studied. The order of activation by monovalent cations was NH₄⁺? K⁺ > Li⁺ > Na⁺. The order of activation by divalent cations was Ca²⁺ > Mn²⁺ = Sr²⁺ > Ba²⁺> Co²⁺ > Mg²⁺ > Zn²⁺. Ethylenediaminetetraacetate inhibits. Activity increased with increasing concentrations of monovalent cations, except for inhibition by increasing concentrations of NH₄⁺ in the presence of Ca²⁺. Adenosine triphosphatase activity was increased by low concentrations of urea and trypsin, but was unaffected by low concentrations of N-ethylmaleimide. For all enzymatic properties where direct comparisons are possible, actomyosin from platelets is unlike that from skeletal muscle, but is similar to that from smooth muscle and non-muscle sources.     

The Bacteroides fragilis NAD-specific Glutamate Dehydrogenase Enzyme is Cell Surface-Associated and Regulated by Peptides at the Protein Level

Bacteroides fragilis has two enzymes with glutamate dehydrogenase (GDH) activity, namely, a dual cofactor NAD(P)H-dependent GDHA, and an NADH-specific GDHB. The presence of two enzymes with the same function is unusual and may play a role in the ability of this organism to survive a variety of environmental conditions. Here we report on the purification and characterisation of the GDHB protein expressed in Escherichia coli from the recombinant plasmid, pGDH15-1 carrying the gdh B gene. The recombinant protein was purified to electrophoretic homogeneity and had a subunit molecular mass of approximately 48 kDa. The temperature and pH activity optima were 38°C and 8.0, respectively, and GDHB enzyme activity was inhibited two-fold by the presence of divalent cations (Ca²⁺, Mg²⁺). The presence of monovalent cations (Na⁺, K⁺) or metabolites (ATP, AMP, ADP or GTP) did not affect enzyme activity. The regulation of GDHB activity was examined at the protein level and evidence of post-translational regulation of the protein in response to peptides but not ammonia was found. Localisation studies using cell fractions of B. fragilis grown under high peptide conditions showed that 79% of GDHB activity was expressed in the membrane fraction. This result was confirmed by immunogold labelling and electron microscopy of B. fragilis cells. It is possible that the GDHB enzyme might play an important role in bacterial survival during invasion of host tissue through its cell-surface location and its regulation via peptides produced by proteases.     

Effect of adsorbed cations on phosphorus uptake by excised roots

Pretreatment of excised roots of Hordeum vulgare, Zea mays, and Glycine max with various salt solutions affected their subsequent rate of phosphorus absorption from 2 × 10⁻⁵m KH₂PO₄. The rate of absorption was greatest for roots pretreated with trivalent cations, intermediate with divalent cations and lowest with monovalent cations. It appeared that the pretreatment involved a rapid exchange reaction at the root surface which was reversible. A 1 min pretreatment was effective for more than 20 min. The acceleration of phosphorus uptake by roots produced by polyvalent cations may be due partly or entirely to a greater reduction in the electrical potential at the root surface or within the pores of the negatively charged cell wall by polyvalent cations than by monovalent cations.     

Cation Penetration through Isolated Leaf Cuticles

The rates of penetration of various cations through isolated apricot Prunus armeniaca L. leaf cuticles were determined. Steady state rates were measured by using a specially constructed flow-through diffusion cell. The penetration rates of the monovalent cations in group IA followed a normal lyotropic series, i.e., CS⁺ ≥ Rb⁺ > K⁺ > Na⁺ > Li⁺. The divalent cations all penetrated through the cuticle more slowly than the monovalent cations. Comparison of the relative values of k (permeability coefficient) and D (diffusion coefficient) indicates that the penetration of ions through isolated cuticles took place by diffusion and was impeded by charge interactions between the solute and charge sites in the penetration pathway. Cuticular penetration rates of K⁺ and H₂O at pH above 9 were of similar magnitude. At pH 5.5 H₂O penetration was not affected but that of K⁺ was greatly reduced. From this observation and from data on cuticle titration and ion adsorption studies, we hypothesize that cuticular pores are lined with a substance (perhaps a protein) which has exposed positively charged sites.     

Effects of Cations on Antimicrobial Activity of Ostricacins-1 and 2 on <Emphasis Type="Italic">E. coli</Emphasis> O157:H7 and <Emphasis Type="Italic">S. aureus</Emphasis> 1056MRSA

Ostricacin-1 and ostricacin-2 (Osp-1 and Osp-2) were β-defensins antimicrobial peptides that were purified from ostrich leukocytes using a cation-exchange column and a semi-prep RP-HPLC column. Both ostricacins were subjected to increased concentrations of monovalent cations (K⁺ and Na⁺) and divalent cations (Ca²⁺ and Mg²⁺) in order to investigate the effect of cations on the activity of these ostricacins on Gram-negative bacteria and Gram-positive bacteria. The radial diffusion assay method showed that both ostricacins were sensitive to the presence of cations. The divalent cations showed more antagonized effect on the activity against Gram-negative bacteria than the monovalent cations, as the ostricacins lost ability to inhibit bacterial growth at very low concentration (5 mM). When viewed in the context of other defensins activity, our data support a hypothesis that defensins’ overall net positive charge determine the sensitivity to cations.     

Ion transport by heart mitochondria. XXII. Spontaneous, energy-linked accumulation of acetate and phosphate salts of monovalent cations

The accumulation of monovalent cations by isolated beef heart mitochondria has been studied by evaluating the efficiency of energy-dependent osmotic swelling. Extensive osmotic swelling occurs spontaneously when isolated heart mitochondria are suspended in 0.1 m acetate or phosphate salts. The swelling and ion uptake depend on either respiration or the presence of exogenous ATP, and the initial rate of swelling is proportional to the initial rate of respiration or ATP hydrolysis, respectively. The efficiency of the reaction varies somewhat from preparation to preparation but approaches a limit of about 2 cations accumulated per pair of electrons traversing a phosphorylation site. All monovalent cations tested support the reaction, but the most efficient energy-dependent swelling occurs with K⁺. Weak acid anions are required for the ion accumulation and swelling and the reaction appears to depend on the amount of free acid available in the suspension. Permeant strong acid anions, such as NO₃⁻, fail to support the swelling reaction in the presence of energy. Valinomycin increases both the amount and the efficiency of ion uptake under these conditions. Mg²⁺ decreases both of these values whereas p-chloromercuriphenyl sulfonate increases both. These responses are discussed in terms of current models of mitochondrial ion transport.     

Nonselective cation channel activated by patch excision from lobster olfactory receptor neurons

Summary A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na⁺, K⁺ and Cs⁺, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage-and concentration-dependent block of Na⁺ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg²⁺ was >1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg²⁺ block of Na^– permeation, and demonstrating the channel's presence in arthropods.     

Binding of Dissolved Strontium by Micrococcus luteus

Resting cells of Micrococcus luteus have been shown to remove strontium (Sr) from dilute aqueous solutions of SrCl₂ at pH 7. Loadings of 25 mg of Sr per g of cell dry weight were achieved by cells exposed to a solution containing 50 ppm (mg/liter) of Sr. Sr binding occurred in the absence of nutrients and did not require metabolic activity. Initial binding was quite rapid (<0.5 h), although a slow, spontaneous release of Sr was observed over time. Sr binding was inhibited in the presence of polyvalent cations but not monovalent cations. Ca and Sr were bound preferentially over all other cations tested. Sr-binding activity was localized on the cell envelope and was sensitive to various chemical and physical pretreatments. Bound Sr was displaced by divalent ions or by H⁺. Other monovalent ions were less effective. Bound Sr was also removed by various chelating agents. It was concluded that Sr binding by M. luteus is a reversible equilibrium process. Both ion exchange mediated by acidic cell surface components and intracellular uptake may be involved in this activity.     

Sperm motility in the horseshoe crab,Limulus polyphemus L: II. Partial characterization of a motility initiating factor from eggs and the effects of inorganic cations on motility initiation

Egg extracts (obtained by washing intact Limulus eggs with either distilled water or artificial seawater, ASW) contain a sperm motility initiating factor (SMI). The SMI is heat stable (withstands boiling to dryness), passes through a dialysis membrane, and is retained by G-10 Sephadex (indicating a molecular weight of less than 700). Qualitative analysis (by X-ray fluorescence spectroscopy) and quantitative analysis (by atomic absorption spectroscopy) of SMI extracts revealed the presence of four divalent cations (Ca, Mg, Ni, and Cu) and one monovalent cation (K) that affect sperm motility. When assayed individually at high concentrations, all of the divalent cations initiate sperm motility and K⁺ inhibits motility initiation by the divalent cations. However, none of the divalent cations are present at concentrations high enough to produce the observed SMI activity, and since K⁺ is present when motility is initiated by SMI, K⁺ must not normally be an inhibitor. Therefore, if inorganic cations are involved in normal sperm motility initiation in Limulus, they are acting in conjunction with some other low molecular weight factor.     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning α-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of “the cation” in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca²⁺, Mg²⁺ and Mn²⁺) and monovalent metal cations (Li⁺, Na⁺, K⁺ and Cs⁺), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation<monovalent amine anesthetic cation<divalent metal cation. We found that organic cations such as the amine anesthetics can also regenerate the proton pump in the bR protein. The inhibition of proton transport in the bR protein by the anesthetic cations was elucidated using the wild type, the E204Q and the D96N mutated bRs. The hydrophobic interaction of the amine anesthetics with the bR protein plays an important part in inhibiting the bR proton pump.     

Mechanism and Role of Divalent Cation Binding of Bacteriorhodopsin

Several observations have already suggested that the carboxyl groups are involved in the association of divalent cations with bacteriorhodopsin (Chang et al., 1985). Here we show that at least part of the protons released from deionized purple membrane (`blue membrane') samples when salt is added are from carboxyl groups. We find that the apparent pK of magnesium binding to purple membrane in the presence of 0.5 mM buffer is 5.85. We suggest this is the pK of the carboxyl groups shifted from their usual pK because of the proton concentrating effect of the large negative surface potential of the purple membrane. Divalent cations may interact with negatively charged sites on the surface of purple membrane through the surface potential and/or through binding either by individual ligands or by conformation-dependent chelation. We find that divalent cations can be released from purple membrane by raising the temperature. Moreover, purple membrane binds only about half as many divalent cations after bleaching. Neither of these operations is expected to decrease the surface potential and thus these experiments suggest that some specific conformation in purple membrane is essential for the binding of a substantial fraction of the divalent cations. Divalent cations in purple membrane can be replaced by monovalent, (Na⁺ and K⁺), or trivalent, (La⁺⁺⁺) cations. Flash photolysis measurements show that the amplitude of the photointermediate, O, is affected by the replacement of the divalent cations by other ions, especially by La⁺⁺⁺. The kinetics of the M photointermediate and light-induced H⁺ uptake are not affected by Na⁺ and K⁺, but they are drastically lengthened by La⁺⁺⁺ substitution, especially at alkaline pHs. We suggest that the surface charge density and thus the surface potential is controlled by divalent cation binding. Removal of the cations (to make deionized blue membrane) or replacement of them (e.g. La⁺⁺⁺-purple membrane) changes the surface potential and hence the proton concentration near the membrane surface. An increase in local proton concentration could cause the protonation of critical carboxyl groups, for example the counter-ion to the protonated Schiff's base, causing the red shift associated with the formation of both deionized and acid blue membrane. Similar explanations based on regulation of the surface proton concentration can explain many other effects associated with the association of different cations with bacteriorhodopsin.