The Effects of pH on the Ionic and Electrical Properties of the Internodal Cells of Chara australis

The effects of pH on the resting and action potentials and onthe fluxes of potassium, sodium, and chloride across the membranesof internodal cells of Chara australis have been investigated. Experiments were carried out in an artificial pond water (A.P.W.)of standard composition: CaCl₂, 01 mM; KCl, 0.1 mM; NaCl, 1.0mM. The resting potential decreased as the pH was lowered from6.5, being depolarized by about 75 mV at pH 4.5. Measurementsof the ion fluxes as a function of pH suggested that this depolarizationwas caused by an increase in the permeability to sodium anda decrease in permeability to potassium at pH 4.5. Action potentialsof constant peak value can be elicited for some time at pH 4.5,but after 20 min or so the cell becomes refractory. All theseeffects on resting and action potentials are fully reversible.We briefly speculate about the mechanism of these pH effects.     

The Electrical Resistance of the Tonoplast of Chara australis

A critical investigation of the method usually used for measuringthe electrical resistance of the tonoplast shows that the valuethus obtained is always higher than the true value.     

Voltage--Current Characteristics of Chara australis During Changes of pH and Exchange of Ca-Mg in the External Medium

Chara australis was the subject of voltage-clamp experimentsin which external calcium, magnesium, and hydrogen were varied.The voltage clamp was applied across the combined plasmalemma,cytoplasm, and tonoplast of internodal cells. Calcium is necessaryin the batching medium for transient currents to occur duringa depolarizing voltage clamp. Magnesium cannot replace calciumas mediator in this function. Lowering the medium pH appearsto affect more than one variable in the membrane system. Weargue that more than one ion is responsible for the transientcurrent; large increases in membrane current of sodium, potassium,and chloride occur.     

Some of the Electrical Characteristics of the Cell Membrane of Chara australis

Measurements of the electrical resistance and potential of thecell membranes of Chara australis have been made for over 60cells. In 17 cases the measurements extended over a period ofa few days for each cell. The most important results of theseobservations and calculations are as follows. The mean valueof the intercellular (between cells) resistance is about 1 kcm². The transcellular (vacuole to external solution) resistanceconsists of two high-resistance layers. The mean value of theelectrical resistance of the outer layer is 7 k cm². The capacityof the transcellular membrane equals about 2.5 µF cm^–2.     

Ionic Activity Gradients across the Surface Membrane of Cytoplasmic Droplets Prepared from Chara australis

The membrane potential and the ionic activity gradients of K⁺and Cl^– across the surface membrane of cytoplasmic dropletsprepared from Chara australis internodal cells, were measuredin high and low ionic strength bathing solutions using liquidion exchange microelectrodes selective for K⁺ and Cl^–.Our results indicate that K⁺ is close to electrochemical equilibriumwhereas Cl^– is not. ¹ Present address: ICI Japan, Palace Hotel Annex Building, Marunouchi,Tokyo, Japan.     

Sustained norepinephrine contraction in the rat portal vein is lost when Ca(2+) is replaced with Sr(2+)

Agonist-induced activation of smoothmuscle involves a rise in intracellular Ca²⁺ concentrationand sensitization of myosin light chain phosphorylation toCa²⁺. Sr²⁺ can enter through Ca²⁺channels, be sequestered and released from sarcoplasmic reticulum, andreplace Ca²⁺ in activation of myosin light chainphosphorylation. Sr²⁺ cannot replace Ca²⁺ infacilitation of agonist-activated Ca²⁺-dependentnonselective cation channels. It is not known whether Sr²⁺can replace Ca²⁺ in small G protein-mediated sensitizationof phosphorylation. To explore mechanisms involved in-receptor-activated contractions in smooth muscle, effects ofreplacing Ca²⁺ with Sr²⁺ were examined in ratportal vein. Norepinephrine (NE) at >3.0 × 10⁷ Min the presence of Ca²⁺ resulted in a strong sustainedcontraction, whereas this sustained component was absent in thepresence of Sr²⁺; only the amplitude of phasic contractionsincreased. Pretreatment with low (~0.05 mM) free Ca²⁺followed by 2.5 mM Sr²⁺ resulted in a sustained componentof the NE response. In -escin-permeabilized preparations,phenylephrine in the presence of GTP or guanosine 5'-O-(3-thiotriphosphate) alone induced sensitization toSr²⁺. In conclusion, a Ca²⁺-regulatedmembrane/channel process is required for the sustained component of NEresponses in rat portal vein. Sensitization alone is not responsiblefor the sustained phase of the NE contraction.

     

Influence of External Osmotic Pressure on Water Permeability and Electrical Conductance of Chara Cell Membrane

Water permeability of the plasma membrane of a Characean internodalcell decreased with an increase in the osmotic pressure of theoutside of the cell, suggesting that the equivalent pore radiusof the water-filled pores becomes smaller with an increase inthe osmotic pressure. In contrast, the apparent membrane resistancedid not increase with an increase in the external osmotic pressure.These facts suggest that ions pass through the membrane mainlyvia pores other than those for bulk water flow. (Received October 22, 1986; Accepted May 22, 1987)     

Partial Purification and Characterization of Aminopeptidase II from Chara australis

Aminopeptidase II, one of the two major aminopeptidases in the giant alga Chara australis, was partially purified. Its molecular weight was estimated to be about 80,000 by gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that it is composed of a single polypeptide with a molecular weight of about 85,000. Aminopeptidase II hydrolyzed alanine-2-naphthylamide more efficiently than the naphthylamides of lysine and proline, and only weakly hydrolyzed the naphthylamides of arginine, phenylalanine, valine, and leucine. The optimal pH for the hydrolysis of alanine-2-naphthylamide was near 7.0. The activity of aminopeptidase II was inhibited by the SH-reagents p-chloromercuribenzoic acid and N-ethylmaleimide and by the metal chelator 1,10-phenanthroline.     

Regulation of the Cytoplasmic pH of Chara corallina: Response to Changes in External pH

Effects of external pH (pH_o) on the cytoplasmic pH (pH_c) ofChara corallina have been measured with the weak acid 5, 5-dimethyloxazolidine-2,4-dione (DMO) following standardized pretreatment of cells insolutions at pHo 4.5, 6.3 and 8.3. Irrespective of pH_c duringpretreatment, pH_o responded to pHo during the experimental periodsof 150–180 min or (in one experiment) 90–110 min.There were increases or decreases of about 0.5 in pHo when cellswere transferred from pH_o 4.5 to 8.3 or vice versa. In the darkpH_c was 0.2–0.3 units lower than the corresponding valuein the light. The results are discussed in relation to the factorsinvolved in the regulation of pH_c in C. corallina, which maybegin to break down below about pH_o4.5, as indicated by relativelylarge decreases in pH_c at low pH_o. Key words: Chara corallina, Cytoplasmic pH, External pH, DMO     

Properties of flavins where the 8-methyl group is replaced by mercapto- residues.

Sulfur functions in position 8 of the flavin nucleus give rise to new modified flavin derivatives, which should prove useful as probes of the flavin binding domains of flavoproteins. Here, we report on some properties of 8-nor-8-alkylmercaptoflavins and 8-nor-8-mercaptoflavin which are readily formed by nucleophilic displacement by alkylmercaptides or sulfide, with 8-nor-8-chloroflavins as starting material. The new flavins are characterized by extensive shifts in spectral properties, with very high extinction coefficients. 8-nor-8-mercaptoriboflavin is easily and reversibly converted to its (-S-S-) dimer. Oxidation of the sulfur group by peracids forms first sulfoxides and then sulfones, in which the characteristic usual flavin spectrum is regained. A comparison of 8-SR-8-nor-flavins with 8-OR-8-nor-flavins (Ghisla, S., and Mayhew, S.G. (1976) Eur. J. Biochem 63, 373-390) indicates that in both classes of compounds, optical properties, ionization constants, and oxidation-reduction potentials follow similar patterns.     

Comparison of copper status in rats when dietary fructose is replaced by either cornstarch or glucose

The purpose of this study was to determine what levels of starch or glucose replacement for fructose in the copper-deficient diet (copper) can minimize the fructose-copper interaction. Experimental diets contained either 100% fructose as the carbohydrate source, or the fructose was partially replaced with 50% starch, 50% glucose, 75% starch, or 75% glucose. Diets were either copper adequate (7-8 ppm) or inadequate (less than 1 ppm). Male weanling rats were fed their respective diet for 5 weeks and then fasted overnight. After decapitation, blood was collected and liver and heart were removed. Plasma copper was significantly reduced and ceruloplasmin was not detected in all copper-deficient groups. Copper deficiency increased plasma cholesterol, as well as heart and liver weight in the glucose groups, but not in the starch groups. Those organ weights were heavier in glucose-copper than starch-copper rats. Erythrocyte copper-zinc-superoxide dismutase activity was greater in starch-copper rats. Erythrocyte copper-zinc-superoxide dismutase activity was greater in starch-copper than glucose-copper rats regardless of carbohydrate amount. Hepatic copper concentration of the group fed starch-copper was twice levels observed in glucose-copper. The 50% glucose rats had lower hepatic copper than the 75% glucose rats. Hepatic copper-zinc-superoxide dismutase activity showed patterns similar to hepatic copper. Cardiac copper was greater in starch-copper than glucose-copper rats. Cardiac copper-zinc-superoxide dismutase activity was equally reduced in all copper-deficient groups. The 50% starch-replaced diet was more effective in minimizing copper deficiency than the 75% glucose-replaced diet. This poorer improvement of copper deficiency by glucose than starch may partially be due to a more severe reduction of food intake in glucose than in starch diets.     

Structural and activity changes in three bioactive anuran peptides when Asp is replaced by isoAsp

The Asp and isoAsp isomers of three bioactive peptides, Crinia angiotensin 11 [APGDRIYHPF(OH)], uperin 1.1 [pEADPNAFYGLM(NH₂)] and citropin 1.1 [GLFDVIKKVASVIGGL(NH₂)] were tested for changes in (i) susceptibility towards proteolytic cleavage, (ii) activity (smooth muscle activity for Crinia angiotensin 11 and uperin 1.1 isomers, and antimicrobial activity for the two isomers of citropin 1.1), and (iii) 3D structures in water, trifluoroethanol-d₃/water (1:1) and DPC micelles as determined by 2D nuclear magnetic resonance spectroscopy. Proteolytic cleavage with trypsin was identical for each pair of Asp/isoAsp isomers. Cleavage with chymotrypsin was the same for the Crinia angiotensin and uperin 1.1 isomeric pairs, but different for the two Asp/isoAsp citropin 1.1 isomers. Chymotrypsin cleaved at Phe3 (adjacent to Asp4) for citropin 1.1, but not at Phe3 (adjacent to isoAsp4) for isoAsp citropin 1.1. The smooth muscle activity of the isoAsp isomer of Crinia angiotensin 11 was less than that of the Asp isomer. The smooth muscle activity of isoAsp3-uperin 1.1 is greater than that of the Asp isomer at low concentration (<10⁻⁹ M) but no different from the Asp isomer at concentrations > 10⁻⁹ M. Citropin 1.1 is a wide-spectrum antibiotic against Gram positive organisms, while the isoAsp isomer is inactive against the test pathogens Staphylococcus aureus and Bacillus subtilis. The observed changes in activity are accompanied by changes in the 3D structures of isomers as determined by 2D nuclear magnetic resonance spectroscopy.     

pH-Sensitive Gating Kinetics of the Maxi-K Channel in the Tonoplast of Chara australis

The most frequently observed K⁺ channel in the tonoplast of Characean giant internodal cells with a large conductance (ca. 170 pS; Lühring, 1986; Laver & Walker, 1987) behaves, although inwardly rectifying, like animal maxi-K channels. This channel is accessible for patch–clamp techniques by preparation of cytoplasmic droplets, where the tonoplast forms the membrane delineating the droplet. Lowering the pH of the bathing solution, that virtually mimicks the vacuolar environment, from an almost neutral level to values below pH 7, induced a significant but reversible decrease in channel activity, whereas channel conductance remained largely unaffected. Acidification (pH 5) on both sides of the membrane decreased open probability from a maximum of 80% to less than 20%. Decreasing pH at the cytosolic side inhibited channel activity cooperatively with a slope of 2.05 and a pK _a 6.56. In addition, low pH at the vacuolar face shifted the activating voltage into a positive direction by almost 100 mV. This is the first report about an effect of extraplasmatic pH on gating of a maxi-K channel. It is suggested that the Chara maxi-K channel possesses an S4-like voltage sensor and negatively charged residues in neighboring transmembrane domains whose S4-stabilizing function may be altered by protonation. It was previously shown that gating kinetics of this channel respond to cytosolic Ca²⁺ (Laver & Walker, 1991). With regard to natural conditions, pH effects are discussed as contributing mainly to channel regulation at the vacuolar membrane face, whereas at the cytosolic side Ca²⁺ affects the channel. An attempt was made to ascribe structural mechanisms to different states of a presumptive gating reaction scheme. Received: 8 May 1998/Revised: 18 September 1998     

Changes in the Membrane Properties of Nodal Cells of Chara braunii Induced by Exposure to Artificial Pond Water

Nodal cells of Chara braunii were exposed to artificial pondwater (APW) for a month or more, with removal of larger surroundingcells. The membrane resistance, resting potential, amplitudeof the action potential, and threshold voltage measured fromthe resting level were examined. Exposure to APW had markedeffects on membrane properties. (Received April 2, 0990; Accepted August 24, 1990)     

Mitochondrial sequestration of BCECF after ester loading in the giant alga Chara australis

Summary. Ratiometric fluorescent dyes are often used to monitor free ion concentrations in vivo, especially in cells that are recalcitrant to transformation with genetically encoded fluorescent markers. Although intracellular dye distributions are often found to be cytosolic, dye localisation has often not been examined in detail. We began exploring the use of BCECF (2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to monitor pH in the giant alga Chara australis and discovered that younger leaf cells could be loaded using the acetoxymethyl ester of BCECF. However, we were puzzled to find in microphotometric measurements that the fluorescence ratio appeared insensitive to manipulations affecting cytosolic pH. Confocal imaging of C. australis cells loaded with BCECF showed an accumulation of the dye in two locations: (1) on the outside of the chloroplasts in irregularly shaped stationary bodies; (2) within 1–1.5 μm structures that moved rapidly with the pericellular cytoplasmic streaming. Together with the streaming cytoplasm, these organelles were rendered stationary with 50 μM cytochalasin D. Rhodamine 123, a mitochondrionspecific dye, highlighted organelles outside of the chloroplasts, similar to those shown by BCECF in location 1. We conclude that in the cytoplasmic compartment, BCECF was sequestered within cytoplasmic mitochondria in immature and fast-growing cells and within the cortical mitochondrial system in older and slowly growing cells. Thus, BCECF-AM is unsuitable for reporting changes in cytosolic pH in C. australis but might be employed in future to study pH changes in the mitochondria. Correspondence: M. J. Beilby, Biophysics, School of Physics, University of New South Wales, Sydney 2052, Australia.     

Evaluation of selective media for Campylobacter isolation when cycloheximide is replaced with amphotericin B

AIMS: Laboratory media for the isolation of Campylobacter usually contain various selective agents, designed to allow these bacteria to grow whilst suppressing that of other organisms. For example, cycloheximide has often been incorporated into Campylobacter media to inhibit the growth of fungi. The production and availability of cycloheximide, however, has recently decreased due to concerns relating to its potential toxicity for mammalian cells and the compound has also become more expensive as a result. An alternative antifungal agent is necessary, and to address this, the effect of using amphotericin B in place of cycloheximide in Campylobacter selective broths and agars was examined. METHODS AND RESULTS: The growth of Campylobacter strains and the suppression of potential competitor organisms in selective broths or on selective agars containing either amphotericin B or cycloheximide was quantified, using pure microbial cultures. The results were then confirmed by testing food and water samples that contained high levels of micro-organisms, including Campylobacter. CONCLUSIONS: The results of this study indicate that amphotericin B is a suitable replacement for cycloheximide in Campylobacter selective media.     

Action of Mono- and Difluorodinitrobenzene on Induced Electrical Modifications by External pH Changes in Nitella

The action of mono- (FNB) or difluorodinitrobenzene (DFNB) onion permeability is mainly attributed to its interactionwithamino groups of the membrane by dinitrophenylation. Nitella cells were dimtrophenylated at pH 7.3 and the membranepotential and electrical resistance were then measured in acidicor basic solutions. No matter what the pH value was, FNB andDFNB induced a depolarization of the membrane potential andcaused a diminution of resistance. However these effects ofFNB and DFNB were more drastic at alkaline pH and in the presenceof a weak concentration of potassium. Neither the addition of0.1 mM calcium nor the substitution of chlorides by nitratesmodified the DFNB effect. These results are compatible withthe assumption that the DFNB binding to the membrane leads toan augmentation of the negative charges of the membrane bringingabout an increased cation conductance and a modification ofthe affinity of a K⁺/H⁺exchange pump. The transient responseof the membrane potential at the time of dinitrophenylationwas used to roughly estimate the total density of amino groupsof the membrane of Nitella.     

Metabolic Conversion of Amino Acids Loaded in the Vacuole of Chara australis Internodal Cells

Vacuoles of internodal cells of Chara australis (or Chara corallina) were loaded with a 10 millimolar amount of various amino acids by a perfusion method and incubated under continuous light. After 20 to 24 hours, the cell sap was collected, and free amino acids in it and the rest of the cell (cytoplasm) were analyzed. The only amino acid metabolized completely was alanine. About 40 to 80% of the aspartic acid, glutamine, serine, and glycine were metabolized, whereas less than 30% of the threonine, asparagine, isoasparagine, isoleucine, phenylalanine, γ-aminobutyric acid, lysine, and arginine were metabolized. The figure for glutamic acid fluctuated between 10 and 100%. The main metabolites of alanine were glutamine, glycine and ammonia, which accumulated in the vacuole. Alanine utilization was not affected by l-methionine-d,l-sulfoximine or azaserine, but was strongly inhibited by aminooxyacetate. The cell extract contained enough alanine aminotransferase activity to account for the rate of alanine metabolism.     

Control of Plasma Membrane Cl- Fluxes in Chara corallina by External Cl- and Light

The efflux of Cl^– at the plasma membrane of Chara wasstudied in relation to two treatments known to affect the flux:that of removal of external Cl^– and of light. It is shownthat although removal of external Cl^– results in a rapidreduction in Cl^– efflux (consistent with a direct effectof external Cl^– on the transport system) the magnitudeof this reduction in the dark is greater than the measured darkinflux. Therefore, in the dark at least, it is proposed that1:1 exchange diffusion cannot account for the trans-stimulationof efflux by external Cl^–. Light induces an inhibition of efflux and a concomitant stimulationof Cl^– influx at 20 °C, but at 10 °C the responsesto light of the two fluxes can be separated temporally. It istherefore suggested that the fluxes are not reciprocally dependenton the same factor which mediates in the light response. Furtherconsiderations show that it is unlikely that the cytoplasmicCl^– concentration mediates in the light response of eitherflux, but that changes in cytoplasmic pH may do so.     

Malate Accumulates in the Vacuole of Chara australis During Uptake of Ammonium From Chloride-free Solution

Internodal cells of Chara australis can accumulate ammoniumto high concentrations (10 to 70 mol m^–3) in their vacuoles.When Cl^– is included in the bathing solution, changesin the cellular concentrations of ammonium, K⁺, Cl^– andNa⁺ have been shown to meet the requirements for electroneutralityand to account for the changes in vacuolar osmotic pressureassociated with ammonium uptake. If accumulation occurs in theabsence of external Cl^–, however; changes in the inorganicions do not meet these criteria. Malate is found in the vacuolesof cells accumulating amine in the absence of external Cl^–and its presence (at 0·5 to 8·5 mol m^–3)allows us to account for electroneutrality and for changes inthe osmotic potential. Key words: Malate, Chara, electroneutrality, ammonium