Catabolite inactivation of phosphoenolpyruvate carboxykinase in spheroplasts from Saccharomyces cerevisiae.

Catabolite inactivation of phosphoenolpyruvate carboxykinase was studied in yeast spheroplasts using 0.9 M mannitol or 0.6 M potassium chloride as the osmotic support. In the presence of potassium chloride the rate of catabolite inactivation was nearly the same as that occurring in intact yeast cells under different conditions of incubation. However, in the presence of mannitol, catabolite inactivation in spheroplasts was prevented. The mannitol inhibition of catabolite inactivation was released by addition of ammonium or phosphate ions. At a concentration of 0.3 M ammonium or 0.06 M phosphate ions, the maximum rate of catabolite inactivation in spheroplasts suspended in mannitol was achieved and was comparable with that observed in spheroplasts incubated in 0.6 M potassium chloride as the osmotic stabilizer. Sodium sulfate (0.04 and 0.4 M) or potassium chloride (0.06 and 0.6 M) did not release the mannitol inhibition of catabolite inactivation in spheroplasts. In intact yeast cells, 0.9 M mannitol, 0.08 M ammonium or 0.1 M phosphate ions did not influence the rate of catabolite inactivation. The nature of the effect of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is discussed.     

Spheroplasts of Torulopsis candida yeasts and their oxidizing activity]

The paper describes the conditions in which the spheroplasts of the yeast Torulopsis candida IBFM-Y-127 with a high respiration rate can be isolated. The preliminary incubation of the cells with SH-reagents has to be carried out in a buffer without an osmotic stabilizer, and the incubation in a medium containing 0.6 M KCL, 0.1 M MgSO4, 0.1 MKH2PO4, pH 5.2. In these conditions the cells are incubated with the enzyme from Helix pomatia during 15 to 20 minutes, and the yield of the spheroplasts is 95 to 100 per cent. The spheroplasts oxidize various substrates (glucose, acetate, ethanol) at the same, or even higher, rate as the intact cells.     

Sporulation of Spheroplasts in Saccharomyces cerevisia

Spheroplasts of Saccharomyces cerevisiae sporulated in 0.1 Mpotassium acetate solution which contained 0.8 M sorbitol ormannitol as the osmotic stabilizer. The appearance of matureasci in both spheroplasts and intact cells was retarded by theaddition of the osmotic stabilizer. Sporulation was repressedmarkedly when 0.6 M KCl was used as the osmotic stabilizer inthe sporulation medium. The germination rate of the spores formedin spheroplasts was 97%. Tetrad analysis showed that meiosiswas normal during the sporulation of spheroplasts. (Received September 5, 1980; Accepted November 29, 1980)     

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

Roles of potassium and chloride ions in cAMP-mediated amylase exocytosis from rat parotid acini.

The roles of potassium and chloride ions in cAMP-mediated amylase exocytosis were studied using intact and saponin-permeabilized parotid acini. Cyclic AMP-evoked amylase release from saponin-permeabilized parotid acini decreased markedly when KCl in the incubation medium was isoosmotically replaced by K-glutamate, NaCl, Na-isothionate, or mannitol. Quinidine and barium, K+ channel blockers, clearly inhibited amylase release from the permeabilized acini, but not from intact ones. The chloride channel blocker DPC (diphenylamine-2-carboxylate) also inhibited amylase release, while DIDS (4,4'-diisothiocyanostilben-2,2'-disulfonate) or bumetanide had little effect, if any, on the exocytosis. Hyperosmolarity with mannitol markedly reduced amylase release from permeabilized acini. These results suggest that potassium and chloride ions play important roles in cAMP-mediated amylase exocytosis, and that these ions act on secretory granules inside the acinar cells.     

Saccharomyces cerevisiae spheroplasts are sensitive to the action of diphtheria toxin.

Diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae but not the intact yeast cells. After 2 h of exposure to ca. 10(-7) M toxin, less than 1% of spheroplasts were able to regenerate into intact cells. The same high levels of toxin inhibited the rate of protein synthesis by more than 90% within 1 h, whereas RNA and DNA synthesis were not inhibited until 4 h or exposure. Both killing and protein synthesis inhibition were dependent on toxin concentration. The nature of the toxin-cell interaction was also studied by using fragments of intact toxin and mutant toxin proteins. Neither toxin fragment A nor CRM45 nor CRM197 affected spheroplasts, but CRM197 and ATP prevented the inhibitory action of intact toxin. These results suggest that toxin acts on S. cerevisiae spheroplasts in much the same manner as it acts on sensitive mammalian cells.     

Intracellular localization of glycolate dehydrogenase in a blue-green alga

Glycolate dehydrogenase activity was detected in cell-free extracts of Oscillatoria sp. prepared by osmotic lysis of spheroplasts in 0.05 m potassium phosphate buffer, pH 7.5, containing 0.3 m mannitol. Most of the enzyme activity was found in a particulate fraction and localized in the photosynthetic lamellae after centrifugation in a discontinuous sucrose density gradient. Enzyme activity was detected in this fraction both in the presence and absence of the artificial electron acceptor 2,6-dichlorophenolindophenol (DPIP) and a low rate of O(2) uptake was detected in this lamellar fraction. Activity was lost from the lamellar fraction by repeated washing or by treatment with 0.005% Triton X-100 and the solubilized enzyme activity was DPIP-dependent. The data indicate that both glycolate dehydrogenase and its natural electron acceptor are bound to the photosynthetic lamellae in vivo. In contrast, catalase activity was found in the soluble cytoplasmic fraction.     

Combination of ammonium nitrate, cerium chloride and potassium chloride salts improves Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum

The frequency of plant transformation can be improved by addition of various chemical into transformation media. In the past, we showed that exposure of tobacco, wheat and triticale explants to ammonium nitrate, cerium and lantanium chloride and potassium chloride resulted in an increase in the frequency of transformation. Here, we tested whether a combination of increased concentrations of the aforementioned salts yielded a higher transformation frequency. We found that exposure to 61.8 mM ammonium nitrate caused a 5.0-fold increase in transformation frequency, whereas exposure to 1.0 μM cerium chloride or 47.0 mM potassium chloride resulted in 1.2- and 2-fold increases, respectively. Exposure to 61.8 mM ammonium nitrate and 1.0 μM cerium chloride led to a 4.8-fold increase in transformation frequency, whereas exposure to 61.8 mM ammonium nitrate and 47.0 mM potassium chloride let to a 5.2-fold increase. Finally, exposure to 61.8 mM ammonium nitrate, 1.0 μM cerium chloride and 47.0 mM potassium chloride produced a 5.1-fold increase. The analysis of the intactness of T-DNA borders showed that plants exposed to ammonium nitrate and a combination of ammonium nitrate with other salts had the more intact right borders and the less intact left borders. The best results were observed when all three salts (ammonium nitrate, potassium chloride and cerium chloride) were used. Thus, we concluded that the addition of cerium chloride and potassium chloride does not substantially improve the transformation rate beyond the improvement observed upon treatment with 61.8 mM ammonium nitrate, but may slightly improve the intactness of T-DNA borders.     

Changes in potassium fluxes in cells of carrot storage tissue related to turgor pressure

The effect of increasing external osmotic pressure on potassium fluxes in aged and fresh-cut discs of Daucus carota L. storage tissue was investigated. An increase of the external osmotic pressure by 5 bars of mannitol solution increased the rate of K⁺ net uptake of aged discs to 180% of their control rate. At 3°C and in 0.1 m M azide, in which a net efflux of potassium was observed, the mannitol treatment caused a reduction in the net efflux. In fresh-cut discs, in which the capability of net influx was rather low and a substantial net release of potassium was noted, the increase in the external osmotic pressure by mannitol caused a 70% inhibition in the net efflux. This effect was also observed at 3°C.
Measurements of separate fluxes confirmed the assumption that the mannitol treatment brought about two distinct effects on K⁺ fluxes: (a) raised the metabolically-dependent influx and (b) lowered the membrane permeability-dependent efflux. When a permeating solute (ethylene glycol) was used instead of mannitol, no effect on K⁺ flux was detectable. Reasons are given for relating the observed changes in K⁺ fluxes to the reduction in turgor pressure of the cells.     

Stability of the Plasma Membrane in Saccharomyces rouxii and Its Relationship to Glucose Tolerance

The stability of spheroplasts from the osmotrophic yeast Saccharomyces rouxii was studied in buffered solutions of mannitol and glucose. The plasma membranes from cells grown in high glucose concentrations were more stable to osmotic lysis than were membranes from cells grown in lower glucose concentrations. Mannitol was a better osmotic stabilizer than glucose, except when the cells were grown in a high glucose concentration. Spheroplasts from a glucose tolerant-deficient mutant were much less stable than the corresponding spheroplasts from the parent strain, especially when suspended in glucose solutions. These results suggest an involvement of the plasma membrane in the glucose-tolerant mechanism of S. rouxii.     

Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase

Phosphotransacetylase (EC 2.3.1.8) was purified 83-fold to a specific activity of 2.5 mmol of acetyl-CoA synthesized per min/mg of protein from Methanosarcina thermophila cultivated on acetate. This rate was 10-fold greater than the rate of acetyl phosphate synthesis. The native enzyme (Mr 42,000-52,000) was a monomer and was not integral to the membrane. Activity was optimum at pH 7.0, and 35-45 degrees C. The enzyme was stable to air and to temperatures up to 70 degrees C, but was inactivated at higher temperatures. Phosphate and sulfate partially protected against heat inactivation. Potassium or ammonium ion concentrations above 10 mM were required for maximum activity of the purified enzyme; the intracellular potassium concentration of M. thermophila approximated 175 mM. Sodium, phosphate, sulfate, and arsenate ions were inhibitory to enzyme activity. Western blots of cell extracts showed that phosphotransacetylase was synthesized in higher quantity in acetate-grown cells than in methanol-grown cells.     

Efflux of potassium ions from cells and spheroplasts of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeast treated with silver and copper ions

Silver ions induce the efflux of potassium from cells of the yeast Saccharomyces cerevisiae but have no such effect on spheroplasts. Copper ions and the natural fungicide 2-O-3-hydroxyhexanoyl-β-D-glucopyranosyl-(1→4)-(6-O-acetyl-β-D-glucopyranosyl-(1→16)-2,15,16-trihydroxyhexadecanoic acid) induce the efflux of potassium ions from both cells and spheroplasts of S. cerevisiae. Silver and copper ions inhibit the activity of the plasma membrane H⁺-ATPase during the treatment of both cells and spheroplasts. It is supposed that the inability of silver ions to stimulate potassium efflux from spheroplasts results from damage to some components of K⁺ transport systems during preparation of spheroplasts.     

Orientation of Membrane Vesicles from Escherichia coli as Detected by Freeze-Cleave Electron Microscopy

The application of freeze-cleave electron microscopy to whole cells of Escherichia coli revealed that the particles exposed on the resulting two inner membrane faces are asymmetrically distributed. This method can therefore be used to determine the orientation of membrane vesicles from E. coli. Membrane vesicles freshly prepared in potassium phosphate buffer (K(+)-vesicles) by osmotic lysis of spheroplasts consisted almost entirely of right-side-out vesicles. Their size suggested that each cell gives rise to one vesicle. When the membrane vesicles were subjected to one cycle of freezing and thawing, the number of inside-out vesicles rose to about 25%. However, due to the small size of most of the inside-out vesicles, these contribute only 2 to 3% of the total membrane surface area of the preparation. The inside-out vesicles appear to arise from infoldings of the membrane of right-side-out vesicles. They also accumulate within the latter, thus producing multivesicular membrane sacs. Na(+)-vesicles (vesicles prepared in sodium phosphate buffer) subjected to freezing and thawing appeared to lose structural rigidity more than did K(+)-vesicles. In contrast to the membrane vesicles prepared by the osmotic lysis of spheroplasts, those obtained by breaking intact cells by a single passage through a French pressure cell were uniformly very small (only 40 to 110 nm in diameter); approximately 60 to 80% were inside-out. To reconcile the polarity of the membrane vesicles with the enzymic activities of such preparations, we propose that "dislocation" of membrane proteins occurs during osmotic lysis of spheroplasts.     

Selective inhibition of the oxidation of ferrous iron or sulfur in Thiobacillus ferrooxidans

The oxidation of either ferrous iron or sulfur by Thiobacillus ferrooxidans was selectively inhibited or controlled by various anions, inhibitors, and osmotic pressure. Iron oxidation was more sensitive than sulfur oxidation to inhibition by chloride, phosphate, and nitrate at low concentrations (below 0.1 M) and also to inhibition by azide and cyanide. Sulfur oxidation was more sensitive than iron oxidation to the inhibitory effect of high osmotic pressure. These differences were evident not only between iron oxidation by iron-grown cells and sulfur oxidation by sulfur-grown cells but also between the iron and sulfur oxidation activities of the same iron-grown cells. Growth experiments with ferrous iron or sulfur as an oxidizable substrate confirmed the higher sensitivity of iron oxidation to inhibition by phosphate, chloride, azide, and cyanide. Sulfur oxidation was actually stimulated by 50 mM phosphate or chloride. Leaching of Fe and Zn from pyrite (FeS(2)) and sphalerite (ZnS) by T. ferrooxidans was differentially affected by phosphate and chloride, which inhibited the solubilization of Fe without significantly affecting the solubilization of Zn.     

Immediate and steady state extracellular ionic fluxes of growing Arabidopsis thaliana root hairs under hyperosmotic and hypoosmotic conditions

Osmotic‐induced shifts in extracellular ionic fluxes at the apex of growing Arabidopsis thaliana L. root hairs were examined using the extracellular self‐referencing (vibrating) ion‐selective probe technique. With either APW 7 (artificial pond water, pH 7) or APW 7 plus 200 m M mannitol/sorbitol, a steady state was reached in which growth rates were the same (0.75 and 0.82 µm min⁻¹) as were the net fluxes of calcium (inward), potassium (outward), and chloride (inward). The outward flux of protons was about 20‐fold larger in APW 7 plus 200 m M mannitol/sorbitol compared to APW 7 ( P = 0.028). Significant changes in ionic fluxes within 5 min of osmotic changes were observed for hyperosmotic treatment: a 7.5‐fold increase in the inward calcium flux ( P = 0.041) and a 1.7‐fold increase in the outward potassium flux ( P = 0.007). Although the change in the calcium flux was consistent with a process of osmotic adjustment (and not a consequence of binding/release from the cell wall), the magnitude of the flux was considerably less than the potassium efflux. Unlike the situation in other organisms where volume regulation explicitly relies upon modulation of ionic fluxes (especially sodium, potassium and chloride), the root hairs may rely on other mechanisms of osmotic regulation, possibly coupled to differences in proton efflux under steady state growth conditions. The root hairs do exhibit an osmotic‐induced electrical signal (R. R. Lew. 1996. Plant Physiol. 112: 1089–1100) which may be a component of initial signal transduction controlling osmotic regulation.     

Mannitol production from glycerol by resting cells of Candida magnoliae

Production of mannitol from glycerol by resting cells of Candida magnoliae under aerobic condition was investigated. The resting cells were suspended in aqueous solution of glycerol in Erlenmeyer flasks and incubated on rotary shaker. The samples were analyzed by ion exclusion–HPLC equipped with refractive index and UV detector. The resting cells of C. magnoliae produced mannitol from fructose, sucrose and glycerol but not from glucose. Addition of yeast extract and/or potassium phosphate to the glycerol solution adversely affected its conversion to mannitol. The conversion of glycerol to mannitol was dependent on oxygen availability. Using resting cells, the yield of mannitol was as high as 45%. This is probably the first report of conversion of glycerol to mannitol by osmophilic yeast.     

The effect of monovalent cations on the catalytic activity of thrombin

The effect of monovalent cations on the catalytic action of thrombin has been examined utilizing a variety of substrates. Sodium chloride noncompetitively inhibited the action of thrombin on α-tosyl-l-arginine methyl ester and α-N-benzoyl-l-arginine-p-nitroanilide. No inhibition was noted when α-N-benzoyl-l-arginine ethyl ester was the substrate. The extent of inhibition was considerably less with either potassium chloride or lithium chloride. The rate of inactivation of thrombin by 1-chloro-3-tosylamido-7-amino-l-2-heptanone was reduced in the presence of sodium ions. The results are interpreted to show a specific effect of sodium ions on the ability of the active-site histidine residue to participate in thrombic catalysis.     

Bioconversion of dethiobiotin to biotin by a cell-free system of a bioYB transformant of Bacillus sphaericus

Abstract Potassium-limited cultures of Candida utilis grown with ammonium chloride as the sole nitrogen source attained a higher dry weight than similar cultures grown with sodium nitrate as the sole nitrogen source. This increase depended on the extracellular ammonia concentration and was not due to accumulation of storage polymers. We conclude that in this yeast ammonium ions can fulfill to some extent the physiological role of potassium ions and that the intracellular concentration of ammonium ions is predominantly determined by the ammonia concentration in the culture medium.     

Differential Effects of Monovalent and Divalent Ions upon the Mode of Action of the Polyene Antibiotic Candicidin

The polyene antibiotic candicidin is a potent membrane active agent, the action of which can be inhibited by the presence of certain ions. The destruction of the selective permeability of yeast membranes by candicidin allows small molecules to leak into the environment. Loss of intracellular potassium ions inhibits yeast glycolysis. This inhibition may be reversed by extracellular concentrations of potassium or ammonium ions. Monovalent ions did not prevent antibiotic absorption or protect yeast growth from the action of the antibiotic. Divalent ions did not protect yeast glycolysis from the action of candicidin, but were able to reduce antibiotic-induced membrane damage and allowed yeast growth in the presence of antibiotic. It is suggested that divalent ions may interact with membrane sterols creating steric hindrance to subsequent candicidin absorption.