Osmotic forces are not critical for Ca2+-induced secretion from permeabilized human neutrophils

In order to examine the role of osmotic forces in degranulation, the effects of solutes and osmolality on granule secretion were explored using both FMLP-stimulated, intact neutrophils and Ca2+-stimulated, permeabilized cells. We employed a HEPES-based buffer system which was supplemented with: a) permeant (KCl or NaCl) or impermeant (Na-isethionate or choline-Cl) ions, or b) permeant (urea) or impermeant (sucrose) uncharged solutes. Intact and permeabilized cells had significantly different solute requirements for degranulation. FMLP-stimulated release from intact cells was supported by NaCl or Na-isethionate greater than KCl greater than choline-Cl or sucrose greater than urea. In contrast, the rank order of Ca2+-stimulated release from permeabilized cells was choline-Cl greater than Na-isethionate, KCl, or NaCl greater than sucrose greater than urea. Hypo-osmotic conditions caused increased levels of background granule release from both intact and permeabilized neutrophils. However, hypo-osmolality inhibited both FMLP-stimulated degranulation from intact cells and Ca2+-induced release from permeabilized neutrophils. While hyperosmotic conditions inhibited stimulated release from intact cells, this inhibition was much less pronounced in permeabilized cells when the granules were directly exposed to these solutions. In fact, hyperosmotic sucrose greatly enhanced Ca2+-induced secretion. Although isolated specific and azurophil granules showed some lytic tendencies in hypo-osmotic buffers, the overall stability of the isolated granules did not indicate that swelling alone could effect degranulation. These results suggest that degranulation in permeabilized cells is neither due to nor driven by simple osmotic forces (under resting or stimulated conditions) and emphasize differences obtained by bathing both the granules and plasma membrane (as opposed to membranes alone) in various solutes.     

Interactions between sarcoplasmic reticulum calcium adenosintriphosphatase and nonionic detergents

The interaction of Triton X-100 and other nonionic detergents with a delipidated preparation of the Ca2+ ATPase from sarcoplasmic reticulum has been studied. Binding of radiolabeled Triton X-100 was determined by column chromatography at 6 degrees C, and two classes of binding sites were observed. Below the critical micelle concentration (cmc), binding of Triton occurred at 35-40 equivalent sites on the delipidated ATPase with a binding constant of 2.7 X 10(4) M-1. Near the cmc cooperative binding of an additional 70 molecules of the detergent was observed. The binding of monomeric Triton X-100 below the cmc was associated with a parallel activation of over half of the ATPase activity, and the remainder of the activity was recovered after the detergent concentration was increased to the cmc. The ability to reactivate ATPase activity was more dependent on the polar poly(oxyethylene) portion of nonionic detergents than on the hydrocarbon portion. Generalizing for all amphiphiles, these results suggest that there are discrete binding sites on the Ca2+ ATPase for phospholipid molecules in the native membrane and that the polar head groups of phospholipids interact more strongly with the protein than the hydrophobic acyl chains. Perturbations in micelle structure were observed for several nonionic detergents by measurement of cis-parinaric acid fluorescence and differential scanning calorimetry, and discontinuities in Arrhenius plots occurred at the transition temperature of the detergent used for reactivation of ATPase activity. It is concluded that both the physiol state of teh micelle and the intrinsic behavior of the ATPase polypeptide affect the temperature dependence of ATPase activity.     

Precipitation of dilute chromatographic samples (ng/ml) containing interfering substances for SDS-PAGE

SDS-PAGE of chromatographic fractions requires prior removal of salts, detergents, denaturants, or organic solvents which may perturb the electrophoretic separation. Likewise, to successfully visualize minute amounts of protein present in chromatographic fractions, they must often be concentrated before analysis by SDS-PAGE. In this study, we used a dye precipitation procedure for simultaneous removal of interfering substances and concentration of dilute samples (ng/ml) before analysis by SDS-PAGE. Nanogram amounts of protein (143 ng) were effectively precipitated with a pyrogallol red-molybdate reagent from commonly used chromatographic buffers containing various interfering solutes or solvents. Proteins were successfully precipitated from solution in the presence of organic solvents (acetonitrile, methanol, 2-propanol), chaotropic agents (6 M urea, 6 M guanidine-HCl), a protein stabilizer (40% sucrose), metal chelators (30 mM EDTA and 30 mM EGTA), or high salt (1.0 M NaCl). Detergents, at concentrations up to twice their critical micelle concentrations, from the nonionic class (Triton X-100, Tween 20) or from the zwitterionic class (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) did not inhibit protein precipitation. Some interference was observed when proteins were precipitated in the presence of ammonium sulfate (0. 5-2.0 M). Proteins did not precipitate in the presence of ionic detergents (SDS and cetyltrimethylammonium bromide). The sensitivity of the combined pyrogallol red-molybdate precipitation/SDS-PAGE procedure is approximately 7 ng. Two other methods of precipitating proteins (trichloroacetic acid and phenol-ether) both exhibited varying degrees of effectiveness, ranging from 714 to 7 ng/ml, in the precipitation of individual proteins. In summary, the pyrogallol red-molybdate protein precipitation procedure facilitates the SDS-PAGE analysis of dilute protein samples (ng/ml) from chromatographic fractions of various compositions. The method is useful for rapid pilot-scale protein fractionation and facilitates the ongoing propensity of researchers to work with minuscule amounts of protein.     

Physico-chemical aspects of solubility of myosin in aqueous media

Solubility of fish (Labio rohita) myosin has been studied at varying temperatures in presence of various inorganic salts like NaCl, KCl, NaBr, Na2SO4, KI, and organic solutes like sucrose and urea. The effect of pH on the solubility has also been studied both in absence and presence of NaCl. Thermal denaturation temperatures of myosin in presence of NaCl, KCl, NaBr and Na2SO4 were found to be 40 degrees, 40 degrees, 45 degrees and 50 degrees C respectively. Thermodynamic parameters like changes in standard free energy (delta G degrees), enthalpy (delta H degrees) and entropy (delta S degrees) for precipitation of myosin from solution phase to gel phase have been evaluated and the physico-chemical aspects have been critically discussed. The average delta G degrees for gel formation varied only between -30 and -40 kJ/mole of myosin, although the nature of solutes, temperature and folding state of protein have been grossly altered. A compensation effect has also been exhibited from the linear plot of average values of delta H degrees against T delta S degrees for various solutes.     

Role of Co-Solute in Biomolecular Stability: Glucose, Urea and the Water Structure

We have studied the action of urea and glucose on the stability of DNAand micelles. We measured the melting temperature of aqueous solutionsof DNA with urea or glucose as a co-solute; we have also measured thechanges in the critical micelle concentrations (cmc) of Sodium DodecylSulfate and Triton X-100 by addition of urea and glucose. Ourexperimental results show that glucose increases the melting temperature ofDNA and decreases the cmc, while urea acts in the contrary direction. The effects of urea and glucose on the stability of DNA and micelles canbe explained by the weakening and enhancement of hydrophobicinteractions, respectively. These effects on hydrophobic interactions arediscussed in this paper.     

Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition are mediated by the packing parameter of phospholipid-detergent systems

The detergent solubilization and reformation of phospholipid vesicles was studied for various detergents. Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition were observed by turbidimetry and cryo-electron microscopy. The first mechanism involves fast solubilization of phospholipids and occurs via open vesicular intermediates. The reverse process, micelle-to-vesicle transition, mimics the vesicle-to-micelle transition. In the second mechanism the solubilization is a slow process that proceeds via micelles that pinch off from closed vesicles. During vesicle reformation, the micelle-to-vesicle transition, a large number of densely packed multilamellar vesicles are formed. The route used, for solubilization and reformation, by a given detergent-phospholipid combination is critically dependent on the overall packing parameter of the detergent-saturated phospholipid membranes. By a change of the overall packing parameter the solubilization and or reformation mechanism could be changed. All five detergents tested fit within the proposed model. With two detergents the mechanism could be changed by changing the phospholipid composition or the medium conditions.     

Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition are mediated by the packing parameter of phospholipid-detergent systems

The detergent solubilization and reformation of phospholipid vesicles was studied for various detergents. Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition were observed by turbidimetry and cryo-electron microscopy. The first mechanism involves fast solubilization of phospholipids and occurs via open vesicular intermediates. The reverse process, micelle-to-vesicle transition, mimics the vesicle-to-micelle transition. In the second mechanism the solubilization is a slow process that proceeds via micelles that pinch off from closed vesicles. During vesicle reformation, the micelle-to-vesicle transition, a large number of densely packed multilamellar vesicles are formed. The route used, for solubilization and reformation, by a given detergent-phospholipid combination is critically dependent on the overall packing parameter of the detergent-saturated phospholipid membranes. By a change of the overall packing parameter the solubilization and or reformation mechanism could be changed. All five detergents tested fit within the proposed model. With two detergents the mechanism could be changed by changing the phospholipid composition or the medium conditions.     

Spin label study of detergents in the region of critical micelle concentration.

A series of spin probes was employed to examine the behavior of the detergent sodium dodecyl sulfate (SDS) at concentrations above and below the critical micelle concentration (cmc). The existence of detergent aggregates below the cmc was evidenced by the appearance of composite electron spin resonance (ESR) spectra for probes that have measurable solubility in water. The spectra were indicative of two probe populations: one in an aqueous environment and another in detergent aggregates. The ESR spectra of probes which are highly insoluble in water exhibited line broadening due to intermolecular spin exchange interactions, indicating that the probes were concentrated in detergent aggregates present below the experimental cmc. The results are discussed in terms of their significance for the study of the mechanisms of micelle formation and for the detection of detergent aggregates at very low concentrations.     

Sucrose monoester micelles size determined by Fluorescence Correlation Spectroscopy (FCS)

One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, R(h). As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured.     

Detergent effects on enzyme activity and solubilization of lipid bilayer membranes

Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturating them. Various concentrations of each detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents must effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.     

Osmotic responses of unicellular blue-green algae (cyanobacteria): changes in cell volume and intracellular solute levels in response to hyperosmotic treatment

Abstract Changes in cell volume and solute content upon hyperosmotic shock have been studied for six unicellular blue-green algae (cyanobacteria): Synechococcus PCC 6301, PCC 6311; Synechocystis PCC 6702, PCC 6714, PCC 6803 and PCC 7008. The extent of change in volume was shown to be dependent upon the solute used to establish the osmotic gradient, with cells in NaCl showing a reduced shrinkage when compared to cells in media containing added sorbitol and sucrose. Uptake of extracellular solutes during hyperosmotic shock was observed in Synechocystis PCC 6714, with maximum accumulation of external solutes in NaCl and minimum solute uptake in sucrose solutions. Conversely, solute loss from the cells (K⁺ and amino acids) was greatest in sucrose-containing media and least in NaCl. The results show that these blue-green algae do not behave as ‘ideal osmometers’ in media of high osmotic strength. It is proposed that short-term changes in plasmalemma permeability in these organisms may be due to transient membrane instability resulting from osmotic imbalance between the cell and its surrounding fluid at the onset of hyperosmotic shock.     

Drinking induced by cellular dehydration in the quail, Coturnix coturnix japonica

1. Drinking was induced in water-replete quail 5-10 min after intravenous injection of hypertonic NaCl (0.69 osmol/l) or sucrose (1.06 osmol/l), but hypertonic urea (2.78 osmol/l) failed to induce drinking. 2. The birds drank approximately the amount required to dilute the injected solutes to isotonicity for each given dose of NaCl or sucrose. 3. The plasma angiotensin II level decreased after injection of 7% NaCl (2.5 osmol/l), but it increased after injection of an equi-osmolar solution of sucrose (65%). 4. Plasma osmolality and Na+ concentration returned quickly to control levels, and then decreased further, after injection of 7% NaCl or 65% sucrose. 5. Blood volume and blood pressure increased immediately after injection of 7% NaCl or 65% sucrose. 6. These results show that drinking is induced after injection of hypertonic solutions exclusively by cellular dehydration, and other regulatory mechanisms for thirst, such as extracellular dehydration and the renin-angiotensin system, are rather inhibitory after injection of hypertonic NaCl.     

Growth,Water Relations,and Accumulation of Organic and Inorganic Solutes in Roots of Maize Seedlings during Salt Stress

Seedlings of maize (Zea mays L. cv Pioneer 3906), hydroponically grown in the dark, were exposed to NaCl either gradually (salt acclimation) or in one step (salt shock). In the salt-acclimation treatment, root extension was indistinguishable from that of unsalinized controls for at least 6 d at concentrations up to 100 mM NaCl. By contrast, salt shock rapidly inhibited extension, followed by a gradual recovery, so that by 24 h extension rates were the same as for controls, even at 150 mM NaCl. Salt shock caused a rapid decrease in root water and solute potentials for the apical zones, and the estimated turgor potential showed only a small decline; similar but more gradual changes occurred with salt acclimation. The 5-bar decrease in root solute potential with salt shock (150 mM NaCl) during the initial 10 min of exposure could not be accounted for by dehydration, indicating that substantial osmotic adjustment occurred rapidly. Changes in concentration of inorganic solutes (Na+, K+, and Cl-) and organic solutes (proline, sucrose, fructose, and glucose) were measured during salt shock. The contribution of these solutes to changes in root solute potential with salinization was estimated.     

Swelling and viscoelastic properties of osmotically stressed elastin

The swelling and viscoelastic properties of purified elastin were studied in aqueous solutions of superswelling agents or osmotic deswelling agents to develop models to study the behavior of elastin at frequencies not easily accessible by direct measurement. Increasing the concentration of any of the deswelling solutes (glucose, sucrose, sodium chloride, ammonium sulphate, dextran, and polyethylene glycol) increased the tensile storage and loss moduli. The viscoelastic behavior was independent of solute when compared on the basis of swelling behavior. The data collected at various solute concentrations at 37°C could be reduced to one master curve, and the master curves for elastin in each of the deswelling solutes were themselves superposable. The ability to reduce the data indicates that dehydration can be used to model elastin's viscoelastic behavior at high frequencies or over short times. The viscoelastic behavior of elastin in the superswelling agents [potassium thiocyanate (KSCN), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)] depended on the solute and was independent of swelling behavior. In KSCN the behavior of elastin seemed to be a continuation of the pattern established by the deswelling agents in that an increase in swelling was accompanied by a decrease in both moduli, and the viscoelastic spectra were reducible to one master curve. In high concentrations of DMSO and EG the spectra were not reducible. KSCN appears a suitable superswelling solute to model elastin's viscoelastic behavior at low frequencies or over long times. © 1996 John Wiley & Sons, Inc.     

The Effect of Water Activity and the aw Controlling Solute on Spore Germination of Bacillus stearothermophilus

The germination of spores of Bacillus stearothermophilus was studied in nutrient broth in relation to the water activity ( a _w) of the medium, the nature of the a _w controlling solutes glycerol, sucrose, KCl, and NaCl, and temperature. Quantitation of germination was based on the change of the phase-bright spore to phase-dark. Activation of spores was by exposure to 100°C/10 min in a medium of the same composition as that used for germination.
Of the four solutes used, sucrose proved most inhibitory to germination, especially in the upper part of the temperature range 38-75°C, glycerol was the most favourable whereas KCl and NaCl, whose effect was almost identical, occupied an intermediate place. The glycerol effect became more pronounced as the a _w of the medium decreased towards 0.960, becoming inhibitory thereafter.
The solute effect on spore germination followed a pattern that related to the class of solute, i.e. electrolyte or non-electrolyte, and its cell penetration characteristics.
Solute penetration during heat activation and germination was considered as the major germination factor and was associated with the osmoregulation mechanism within the spore proposed recently as the basis of spore dormancy and resistance.     

Accumulation of carbohydrate by Escherichia coli B/r/1 during growth at low water activity

The carbohydrate content of Escherichia coli B/r/1, grown in a glucose or arabinose-limited salts medium in a chemostat, increased by a factor of 2–4 when the water activity (a_w) of the medium was reduced to 0.986 by addition of NaCl, KCl or sucrose. The biomass decreased by 30–45%. The sucrose system resulted in the lowest biomass and carbohydrate content. The monosaccharide part of the accumulated carbohydrate consisted of glucose or glucose and arabinose in the cultures fed glucose and arabinose, respectively, and accounted for 50% or more of the total carbohydrate in the NaCl and KCl systems and 16.79% in the sucrose system. In addition, the K⁺ content depended on the solute and related inversely to the monosaccharide content, being highest in the sucrose system. The combined molarity of the monosaccharide and K⁺ was deduced to be far in excess of that required for osmotic equilibration of the cultures, especially in the sucrose system. These observations are discussed in the context of osmoregulation, the effects of solutes on glucose metabolism and the morphological changes that occur in cultures at low a_w.     

Osmoregulation in the Avena Coleoptile : CONTROL OF SOLUTE UPTAKE IN PEELED SECTIONS

Peeled Avena sativa coleoptile sections (i.e. sections from which the epidermis has been removed) have been used to study the control of solute uptake under conditions where the uptake is not limited by the cuticular barrier. In the presence of 2% sucrose, auxin enhances the rate at which the total osmotic solutes increase, but this appears to be a response to the increased growth rate, inasmuch as the auxin effect is eliminated when growth is inhibited osmotically. When sections are incubated in sucrose or in 20 millimolar NaCl, the osmotic concentration increases until a plateau is reached after 8 to 24 hours. Auxin has no effect on the initial rate of increase in osmotic concentration but causes the osmotic concentration to reach a plateau earlier and at a lower osmotic conentration value. This difference in steady-state osmotic concentration is, in part, a response to auxin itself, as it persists when auxin-induced growth is inhibited osmotically. The upper limit for osmotic concentration does not appear to be determined by the turgor pressure, inasmuch as a combination of sucrose and NaCl gave a higher plateau osmotic concentration than did either solute alone. We suggest that the rate of solute uptake is determined by the availability of absorbable solutes and by the surface area exposed to the solutes. Each absorbable solute reaches a maximum internal concentration independent of other absorbable solutes; the steady-state osmotic concentration is simply the sum of these individual internal concentrations.     

Solutes modify a conformational transition in a membrane transport protein

The bacterial outer-membrane vitamin B₁₂ transporter, BtuB, undergoes a dramatic order-to-disorder transition in its N-terminal energy-coupling motif (Ton box) upon substrate binding. Here, site-directed spin labeling (SDSL) is used to show that a range of solutes prevents this conformational change when ligand is bound to BtuB, resulting in a more ordered Ton box structure. For each solute examined, the data indicate that solutes effectively block this conformational transition through an osmotic mechanism. The molecular weight dependence of this solute effect has been examined for a series of polyethylene glycols, and a sharp molecular weight cutoff is observed. This cutoff indicates that solutes are preferentially excluded from a cavity within the protein as well as the protein surface. Furthermore, the sensitivity of the conformational change to solution osmolality is consistent with a structural model predicted by SDSL. When the Ton box is unfolded by detergents or mutations (rather than by ligand binding), solutes, such as polyethylene glycols and salts, also induce a more structured compacted conformation. These results suggest that conformational changes in this class of outer membrane transporters, which involve modest energy differences and changes in hydration, may be modulated by a range of solutes, including solutes typically used in protein crystallization.     

Effects of accumulation of 3-O-methylglucose on levels of endogenous osmotic solutes in Chlorella emersonii

Abstract. Regulation of the concentration of osmotic solutes was studied in Chlorella emersonii grown at external osmotic pressures (II) ranging between 0.08 and 1.64MPa. NaCl was used as osmoticum. The total solute content of the cells was manipulated by applying 2 mol m⁻³ 3- O -methylglucose (MG), which was not metabolized, and accumulated at concentrations ranging between 60 and 230 mol m⁻³ within 4 h after its addition to the medium. Methylglucose uptake resulted in decreases in concentrations of proline and sucrose, the two solutes mainly responsible for osmotic adaptation of C. emersonii to high external II. The responses were consistent with the hypothesis that proline and sucrose concentrations are controlled by a system of osmotic regulation, with turgor and/or volume as a primary signal. Short-term experiments showed that even very small increases in turgor and/or volume, due to accumulation of methylglucose, resulted in large decreases in proline and sucrose. Over the first 30-60 min the total solute concentration in the cells increased by at most 15 osmol m⁻³ which would represent an increase in turgor pressure of at most 0.04 M Pa. Yet, the decreases in proline and sucrose were as fast as those in cells exposed to a sudden decrease of 0.25 MPa in external II, when the turgor pressure would have increased by at least 0.15 MPa. High concentrations of methylglucose in cells grown at high II did not affect the rapid synthesis of proline and sucrose which started when the cells were transferred to yet higher II. Thus, methylglucose had no direct effects on proline and sucrose metabolism, and it has been assumed that it acted solely as an inert osmotic solute within the cell.     

Erythroid spectrin in miceller detergents

We have studied the interaction of spectrin, the major protein of the erythrocyte cytoskeleton, with four commonly used detergents at concentrations above their critical miceller concentrations (cmc). Fluorescence spectroscopic studies on the emission intensity, steady state polarization, quenching with acrylamide, and time-resolved fluorescence measurements were done with spectrin in anionic detergents, e.g., SDS, deoxycholate, and nonionic detergents, e.g., Triton-X-100 and octylglucoside at concentrations double their respective cmc's. The spectrin-detergent complexes in all four systems have been characterized by far-UV CD and measurements on tryptophan fluorescence in combination with fluorescence of the extrinsic probe, pyrene. Tryptophan fluorescence studies revealed quaternary structural changes due to unzipping of the spectrin subunits in Triton-X-100 without complete dissociation. Both Triton-X-100 and SDS were found to partially denature spectrin indicated by the far-UV CD. Octylglucoside and deoxycholate are shown to have the least structural perturbations on the cytoskeletal protein, rationalizing the use of octylglucoside, in particular and also deoxycholate to be the most effective in preparing cytoskeletal fractions from erythrocytes rather than the Triton-X-100 that has long been used for preparing the Triton shells.