A quantitative correlation between the kinetics of solutes and water translocation in Liver Mitochondria

Summary A procedure is described for the calculation of solute fluxes in mitochondria from absorbance measurements. The procedure assumes that mitochondria behave as osmometers and that they are always at osmotic equilibrium.The rates and amounts of K⁺ translocation have been calculated simultaneously, with the photometric procedure and electrometrically, during passive, K⁺ efflux coupled to Ca⁺⁺ uptake and during active K⁺ uptake and passive K⁺ release coupled with anion translocation. Good agreement has been found between the two sets of measurements. The data are compatible with the concept that the energy-linked, ion translocation-coupled, mitochondrial swelling is osmotic in nature. It is concluded that the changes of absorbance are quantitatively related to changes in the inner volume and therefore the photometric procedure can be used to calculate, ion fluxes of osmotically active species under various circumstances.     

Optical changes in unilamellar vesicles experiencing osmotic stress.

Membrane properties that vary as a result of isotropic and transmembrane osmolality variations (osmotic stress) are of considerable relevance to mechanisms such as osmoregulation, in which a biological system "senses" and responds to changes in the osmotic environment. In this paper the light-scattering behavior of a model system consisting of large unilamellar vesicles of dioleoyl phosphatidyl glycerol (DOPG) is examined as a function of their osmotic environment. Osmotic downshifts lead to marked reductions in the scattered intensity, whereas osmotic upshifts lead to strong intensity increases. It is shown that these changes in the scattering intensity involve changes in the refractive index of the membrane bilayer that result from an alteration in the extent of hydration and/or the phospholipid packing density. By considering the energetics of osmotically stressed vesicles, and from explicit analysis of the Rayleigh-Gans-Debye scattering factors for spherical and ellipsoidal shells, we quantitatively demonstrate that although changes in vesicle volume and shape can arise in response to the imposition of osmotic stress, these factors alone cannot account for the observed changes in scattered intensity.     

Reduced osmotic potential inhibition of photosynthesis : site-specific effects of osmotically induced stromal acidification

The effects of reduced reaction medium osmotic potential (0.67 molar sorbitol as compared to a control treatment with 0.33 molar sorbitol) on the enzymic steps of the photosynthetic carbon reduction cycle were investigated using isolated spinach (Spinacia oleracea L. var Longstanding Bloomsdale) chloroplasts. Reversal of reduced osmotic potential inhibition of photosynthetic rates by a stromal alkalating agent (NH₄Cl) was associated with specific steps of the cycle. Low osmotic potential induced stromal acidification was found to be facilitated by osmotically induced chloroplast shrinkage. However, the action of the alkalating agent was found not to be associated with reversal of osmotically induced morphological changes of the stromal compartment.

Labeled metabolite analyses indicated that the osmotic stress treatment caused the substrate for fructose 1,6-bisphosphatase (FBPase) to build up in the absence of NH₄Cl, and the substrate for phosphoribulokinase to increase in the presence of NH₄Cl. These data were interpreted as indicating that the most severe effect of osmotic stress on photosynthesis is at the site of FBPase, and that this inhibition is mediated by osmotically induced stromal acidification. Phosphoribulokinase activity inhibition at the low osmotic potential treatment was apparently less severe and not mediated by stromal acidification. A third site of osmotic inhibition, which was reversed by NH₄Cl, and therefore was assumed to be mediated by stromal acidification, was at the step of ribulose 1,5-bisphosphate carboxylase.

Additions of NH₄Cl also enhanced the activity of the pH-insensitive phase of the photosynthetic carbon reduction cycle, 3-phosphoglyceric acid reduction, at the stress treatment. This effect was thought to be mediated by the removal of the block at FBPase. A model was proposed to outline the relative severity of osmotic stress effects at various sites of the photosynthetic carbon reduction cycle.

     

Solutes in the free space of growing stem tissues

The concentration of osmotically active solutes in the cell wall free space of young stem tissues was studied using a variety of extraction methods. When the intercellular air spaces of etiolated pea (Pisum sativum L.) internodes were perfused with distilled H₂O, the resulting solution contained a solute concentration of about 70 milliosmoles per kilogram. A second procedure involving vacuum infiltration of segments followed by centrifugation to collect the free space solution gave similar results. Apical stem segments yielded free space extracts about twice as concentrated as those from basal portions of the stem. After correcting for dilution of the free space solution by the infiltrated water, the osmotic pressure of the undiluted free space in pea stem tissue was estimated to be 2.9 bars for apical segments, 1.8 bars for basal regions. These values may be somewhat overestimated due to solute efflux from intracellular pools during the extraction procedure. Similar results were obtained for stem regions of etiolated soybean (Glycine max [L.] Merr.) and cucumber (Cucumis sativus L.) seedlings.

From measurements of the electrical conductivity and refractive index of free space extracts before and after ashing, it appears that 25% of the solutes are inorganic electrolytes and 75% are organic nonelectrolytes with an average size similar to that of glucose.

A significant osmotic pressure in the wall space offers an explanation for the frequent observation that nontranspiring plants have negative water potentials. Calculations of hydraulic resistance from water potential data must take into account solutes in the free space, else `apparent,' but unreal, changes in resistance may be calculated.

     

Osmotic significance of glycerol accumulation in exponentially growing yeasts.

Natural-abundance 13C-nuclear magnetic resonance spectroscopy has shown glycerol to be the major osmotically significant low-molecular-weight solute in exponentially growing, salt-stressed cells of the yeasts Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Debaromyces hansenii. Measurement of the intracellular nonosmotic volume (i.e., the fraction of the cell that is osmotically unresponsive) by using the Boyle-van't Hoff relationship (for nonturgid cells, the osmotic volume is directly proportional to the reciprocal of the external osmotic pressure) showed that the nonosmotic volume represented up to 53% of the total cell volume; the highest values were recorded in media with maximum added NaCl. Determinations of intracellular glycerol levels with respect to cell osmotic volumes showed that increases in intracellular glycerol may counterbalance up to 95% of the external osmotic pressure due to added NaCl. The lack of other organic osmotica in 13C-nuclear magnetic resonance spectra indicates that inorganic ions may constitute the remaining component of intracellular osmotic pressure.     

Osmotic significance of glycerol accumulation in exponentially growing yeasts

Natural-abundance 13C-nuclear magnetic resonance spectroscopy has shown glycerol to be the major osmotically significant low-molecular-weight solute in exponentially growing, salt-stressed cells of the yeasts Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Debaromyces hansenii. Measurement of the intracellular nonosmotic volume (i.e., the fraction of the cell that is osmotically unresponsive) by using the Boyle-van't Hoff relationship (for nonturgid cells, the osmotic volume is directly proportional to the reciprocal of the external osmotic pressure) showed that the nonosmotic volume represented up to 53% of the total cell volume; the highest values were recorded in media with maximum added NaCl. Determinations of intracellular glycerol levels with respect to cell osmotic volumes showed that increases in intracellular glycerol may counterbalance up to 95% of the external osmotic pressure due to added NaCl. The lack of other organic osmotica in 13C-nuclear magnetic resonance spectra indicates that inorganic ions may constitute the remaining component of intracellular osmotic pressure.     

Differential Light Scattering Measurements of Heat-Treated Bacteria

Effects of heat on diameter, size distribution, and refractive index of Staphylococcus epidermidis suspensions were determined accurately by computer analysis of differential light scattering data.     

Intermembrane electron transport in the absence of added water-soluble carriers

Electron transport from untreated to mersalyzed microsomal vesicles at the level of NADH-cytochrome b₅ reductase or cytochrome b₅ has been demonstrated in the absence of added water-soluble electron carriers. A similar effect was shown in the systems “intact mitochondria — mersalyzed microsomes” and “mersalyzed mitochondria— untreated microsomes”. No measurable electron transport between intact and mersalyzed particles of inner mitochondrial membrane was found. The obtained data suggest that the capability to carry out intermembrane electron transfer is specific for NADH-cytochrome b₅ reductase and/or cytochrome b₅, localized in microsomal and outer mitochondrial membranes.     

Light scattering turbidity changes as a measure of the kinetics of Ca2+-promoted aggregation of chromaffin granule membrane ghosts

Changes in turbidity seen when chromaffin granule membrane ghosts are aggregated by Ca²⁺ can be modelled as dimerization of hollow spheres using Rayleigh-Gans-Debye light-scattering theory. The experimental changes agree well with the calculations. Thus, if shape or refractive index changes produced by osmotic perturbation, ion uptake, etc. can be excluded, turbidity readings can be used to follow the progress of the aggregation reaction of storage vesicles and other small particles or macromolecules.     

Heterolysosome formation in mouse liver

When formaldehyde-treated ¹³¹I-albumin was injected into mice, the total liver radioactivity did not change significantly from 5 minutes to 60 minutes after injection. There was a progressive increase with time in the amount of radioactivity associated with liver particles which could be released by osmotic shock; the quantity of material tightly bound to particles, but not releasable by osmotic shock, did not change. At five minutes after injection the liver particles did not release acid-soluble radioactivity into the medium when incubated at 37°. These particles contain the injected protein in osmotically releasable form not associated with proteolytic enzymes and therefore correspond to phagosomes. At 10, 30 or 60 minutes after injection, the particles degraded the protein at similar rates but the activity ceased after 90 minutes incubation when only 50 to 60% of the osmotically releasable material was hydrolyzed. This cessation of activity was shown to be due to a thermal disruption of the particles during incubation.     

Products of Proline Catabolism Can Induce Osmotically Regulated Genes in Rice

Many plants accumulate high levels of free proline (Pro) in response to osmotic stress. This imino acid is widely believed to function as a protector or stabilizer of enzymes or membrane structures that are sensitive to dehydration or ionically induced damage. The present study provides evidence that the synthesis of Pro may have an additional effect. We found that intermediates in Pro biosynthesis and catabolism such as glutamine and Δ¹-pyrroline-5-carboxylic acid (P5C) can increase the expression of several osmotically regulated genes in rice (Oryza sativa L.), including salT and dhn4. One millimolar P5C or its analog, 3,4-dehydroproline, produced a greater effect on gene expression than 1 mm l-Pro or 75 mm NaCl. These chemicals did not induce hsp70, S-adenosylmethionine synthetase, or another osmotically induced gene, Em, to any significant extent. Unlike NaCl, gene induction by P5C did not depend on the normal levels of either de novo protein synthesis or respiration, and did not raise abscisic acid levels significantly. P5C- and 3,4-dehydroproline-treated plants consumed less O₂, had reduced NADPH levels, had increased NADH levels, and accumulated many osmolytes associated with osmotically stressed rice. These experiments indicate that osmotically induced increases in the concentrations of one or more intermediates in Pro metabolism could be influencing some of the characteristic responses to osmotic stress.     

THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.     

Sucrose Is a Nonaccumulated Osmoprotectant in Sinorhizobium meliloti

Intracellular accumulation of sucrose in response to lowered water activity seems to occur only in photosynthetic organisms. Here we demonstrate, for the first time, the potent ability of this common sugar, supplied exogenously, to reduce growth inhibition of Sinorhizobium meliloti cells in media of inhibitory osmolarity. Independently of the nature of the growth substrates and the osmotic agent, sucrose appears particularly efficient in promoting the recovery of cytoplasmic volume after plasmolysis. Surprisingly, sucrose is not accumulated by the bacteria at an osmotically efficient level. Instead, it strongly stimulates the accumulation of the main endogenous osmolytes glutamate and N-acetylglutaminylglutamine amide (NAGGN). Examining cell volume changes during the hyperosmotic treatment, we found a close correlation between the enhancement of the osmotically active solute pool and the increase in cell volume. Sucrose shares several features with ectoine, another nonaccumulated osmoprotectant for S. meliloti. Overall, osmoregulation in S. meliloti appears to be strongly divergent from that in most bacteria.     

Water balance in Collembola and its relation to habitat selection: Water content,haemolymph osmotic pressure and transpiration during an instar

In the two collembolan species Orchesella cincta and Tomocerus minor the water content, haemolymph osmotic pressure and transpiration rate fluctuate with the feeding rhythm during each instar. The changes in water content, however, are due to changes in dry weight, because the absolute water weight stays constant during the instar.The intake of food is probably the cause of the increase in haemolymph osmotic pressure. Increase of osmotically active substances in the blood and/or blood volume reduction may be responsible for the rise in osmotic pressure. This change in osmotic pressure in turn may affect the responsiveness of the animals to water as well as their feeding behaviour.Changes in the epicuticle and in epidermal cell membranes may cause changes in the rate of transpiration. The high rate observed during ecdysis and during the mid-instar may explain the behaviour of the animals in varied water conditions.Dehydration during the instar causes an equivalent rise in osmotic pressure for both Tomocerus minor and Orchesella cincta. The water loss appears to involve the haemolymph. The physiological state of the animal does not influence the rise in osmotic pressure. There are no signs of any osmoregulation in the two species.     

Cell volume dependence of 1H spin-echo NMR signals in human erythrocyte suspensions: The influence of in situ field gradients

The ¹H spin-echo NMR signal amplitudes and intensities of low molecular weight solutes in the cytoplasm and extracellular fluid of suspensions of human erythrocytes were shown to depend on the osmotic pressure of the media. At low osmotic pressure (220 mosM/kg) freeze-thaw lysis of the cells resulted in signal enhancement which was greatest for extracellular molecules, but both intra- and extracellular species were almost equally enhanced at 580 mosM/kg. This effect is due to field gradients formed at cell boundaries as a result of differences in magnetic susceptibility between the medium and the cytoplasm. T₂ values measured using the Carr-Purcell-Meiboom-Gill pulse sequence, with τ = 0.0003 s, depended little on cell volume and absolute changes in volume magnetic susceptibility were also small. The mean field gradients, calculated from data obtained on cell suspensions at different osmotic pressures, were in the range 0.25–1.98 G/cm and 0.89–2.09 G/cm for intra- and extracellular compartments, respectively. The maintenance of isotonicity of the extracellular fluid during metabolic studies of cell suspensions is important in order to avoid artefacts in the determination of metabolite concentrations when using the spin-echo technique. Conversely it may be possible to perform transport measurements using spin-echo NMR to monitor the cell volume changes which occur during the transmembrane migration of molecules.     

ACTION OF TRITON X-100 ON CHLOROPLAST MEMBRANES : Mechanisms of Structural and Functional Disruption

Addition of Triton X-100 to chloroplast suspensions to a final concentration of 100–200 µM causes an approximate tripling of chloroplast volume and complete inhibition of light-induced conformational changes, light-dependent hydrogen ion transport, and photophosphorylation. Electron microscopic studies show that chloroplasts treated in this manner manifest extensive swelling in the form of vesicles within their inner membrane structure. Triton was adsorbed to chloroplast membranes in a manner suggesting a partition between the membrane phase and the suspending medium, rather than a strong, irreversible binding. This adsorption results in the production of pores through which ions may freely pass, and it is suggested that the inhibition of conformational changes, hydrogen ion transport, and photophosphorylation by Triton is due to an inability of treated chloroplast membranes to maintain a light-dependent pH gradient. The observed swelling is due to water influx in response to a fixed, osmotically active species within the chloroplasts, after ionic equilibrium has occurred. This is supported by the fact that chloroplasts will shrink upon Triton addition if a nonpenetrating, osmotically active material such as dextran or polyvinylpyrrolidone is present externally in sufficient concentration (>0.1 mM) to offset the osmotic activity of the internal species.     

STUDIES ON OSMOTIC EQUILIBRIUM AND ON THE KINETICS OF OSMOSIS IN LIVING CELLS BY A DIFFRACTION METHOD

1. Osmotic equilibrium and kinetics of osmosis of living cells (unfertilized eggs of Arbacia punctulata) have been studied by a diffraction method. This method consists of illuminating a suspension of cells by parallel monochromatic light and measuring, by means of telescope and scale, the angular dimensions of the resulting diffraction pattern from which the average volume of the cells may be computed. The method is far less laborious and possesses several advantages over direct measurement of individual cells. The average size of a large number of cells is obtained from a single measurement of the diffraction pattern and thus individual variability is averaged out. The observations can be made at intervals of a few seconds, permitting changes in volume to be followed satisfactorily. During the measurements the cells are in suspension and are constantly stirred. 2. Volumes of cells in equilibrium with solutions of different osmotic pressure have been determined. In agreement with our previous experiments, based upon direct microscope measurements, we have confirmed the applicability of the law of Boyle-van''t Hoff to these cells; that is to say, the product of volume and pressure has been found to be approximately constant if allowance be made for the volume of osmotically inactive material of the cell contents. The volume of osmotically inactive material was found to be, on the average, 12 per cent of the initial cell volume; in eggs from different animals this value ranged from 6 to 20 per cent. 3. Permeability to water of the Arbacia egg has been found to average, at 22°C., 0.106 cubic micra of water per square micron of cell surface, per minute, per atmosphere of difference in osmotic pressure. 4. Permeability to ethylene glycol has been found to average, at 24°C., 4.0 x 10^–15 mols, per square micron of cell surface, per minute, for a concentration difference of 1 mol per liter. This is in agreement with the values reported by Stewart and Jacobs.     

Osmoregulation in Rhizobium meliloti: Production of Glutamic Acid in Response to Osmotic Stress

Rhizobium meliloti, like many other bacteria, accumulates high levels of glutamic acid when osmotically stressed. The effect was found to be proportional to the osmolarity of the growth medium. NaCl, KCI, sucrose, and polyethylene glycol elicited this response. The intracellular levels of glutamate and K⁺ began to increase immediately when cells were shifted to high-osmolarity medium. Antibiotics that inhibit protein synthesis did not affect this increase in glutamate production. Cells growing in conventional media at any stage in the growth cycle could be suspended in medium causing osmotic stress and excess glutamate accumulated. The excess glutamate did not appear to be excreted, and the intracellular level eventually returned to normal when osmotically stressed cells were suspended in low-osmolarity medium. A glt mutant lacking glutamate synthase and auxotrophic for glutamate accumulated excess glutamate in response to osmotic stress. Addition of isoleucine, glutamine, proline, or arginine stimulated glutamate accumulation to wild-type levels when the mutant cells were suspended in minimal medium with NaCl to cause osmotic stress. In both wild-type and mutant cells, inhibitors of transaminase activity, including azaserine and aminooxyacetate, reduced glutamate levels. The results suggest that the excess glutamate made in response to osmotic stress is derived from degradation of amino acids and transamination of 2-ketoglutarate.     

Tonoplast fine structure and osmotic regulation in Porphyra umbilicalis

In Porphyra, an intertidal red alga, the fine structure of the tonoplast was studied by freeze-fracture electron microscopy. It was shown that density and size of intramembraneous particles on the protoplasmic fracture face vary with external osmotic potential. The frequency of particles grouped in size classes (calculated per cell) increases with increasing osmotic stress and shows a maximum in 3 to 4 x artificial seawater medium ASP₁₂. It is concluded that the intensity of tonoplast transport, which probably is enhanced with increasing osmotic stress from 1 to 4 x media, is most likely correlated with a change in membrane fine structure of the tonoplast.