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.     

Regulation of internal solute concentrations of marine Vibrio alginolyticus in response to external NaCl concentration.

Slightly halophilic marine Vibrio alginolyticus grown in the range of NaCl from 0.2 to 1.5 M maintained the total internal solute concentration always higher than the external medium by about 0.25 osM. The concentrations of macromolecules such as DNA, RNA, and protein were little affected by the increase in medium NaCl. The internal K+ concentration was kept to about 400 mM in the range of medium NaCl from 0.4 to 0.8 M; it rose to 510 mM when the bacterium was grown in 1.5 M NaCl, indicating that K+ increased only slightly in response to the large increase in medium NaCl. Thus, in contrast to the case of nonhalophilic and extremely halophilic bacteria, K+ was unlikely to act as a major component to regulate the internal solute concentration of marine V. alginolyticus. The internal Na+ and Cl- concentrations were maintained always lower than those in the growth medium, but they increased in response to the increase in medium NaCl. The concentration of internal Na+ was close to that of K+ at the concentration of medium NaCl that supports the optimal growth of this organism. The total amino acid content of V. alginolyticus increased from 76 to 413 mM by the increase in medium NaCl from 0.2 to 1.5 M. The concentrations of glutamic acid and prolined were 254 and 72 mM, respectively, when grown in 1.5 M NaCl. These results indicated that Na+, Cl- and amino acids, especially glutamic acid and proline, contributed to the regulation of internal solute concentration of V. alginolyticus in response to the increased external NaCl.     

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.     

Free amino acid dynamics in marine methanogens. beta-Amino acids as compatible solutes.

Methanogenic archaebacteria respond to osmotic stress by accumulating a series of organic molecules which function as compatible solutes. In two strains of marine methanogenic archaebacteria, Methanogenium cariaci and Methanococcus thermolithotrophicus, four key organic solutes are observed: L-alpha-glutamate, beta-glutamate, N epsilon-acetyl-beta-lysine, and betaine. The first three of these are synthesized de novo; betaine is transported into the Mg. cariaci cells from the medium. Mesophilic Mg. cariaci will preferentially transport betaine from the extracellular medium if it is present to counterbalance the external NaCl. In its absence it synthesizes N epsilon-acetyl-beta-lysine as the dominant osmolyte. This zwitterionic compound occurs at levels in Mg. cariaci which are considerably greater (based on mumol/mg of protein) than in Mc. thermolithotrophicus grown in media of the same ionic strength. Intracellular potassium ion concentrations, determined by 39K NMR spectroscopy and atomic absorption, differ significantly in the two cells. In Mc. thermolithotrophicus, intracellular K+ is balanced by the total concentration of anionic amino acid species, glutamate, and beta-glutamate. Turnover of the organic solutes has been monitored using 13C-pulse/12C-chase, and 15N-pulse/14N-chase experiments. Both beta-amino acids exhibit slower turnover rates when compared to L-alpha-glutamate or aspartate, consistent with their roles as compatible solutes. Biosynthetic information for the beta-amino acids is also provided by 13C-labeling experiments. beta-Glutamate shows a lag in 13C uptake from 13CO2, indicative of its biosynthesis from a precursor (probably a macromolecule) not in equilibrium with the soluble L-alpha-glutamate pool. Confirmation of a novel route for beta-glutamate synthesis and the production of the beta-lysine moiety from the diaminopimelate pathway is deduced from [13C2]acetate labeling patterns.     

Compatible Solutes in the Thermophilic Bacteria Rhodothermus marinus and "Thermus thermophilus"

(sup13)C nuclear magnetic resonance spectroscopy and (sup1)H nuclear magnetic resonance spectroscopy were used to identify and quantify the organic solutes of several strains of halophilic or halotolerant thermophilic bacteria. Two strains of Rhodothermus marinus and four strains of "Thermus thermophilus" grown in complex medium containing NaCl were examined. 2-O-Mannosylglycerate was a major compatible solute in all strains: the Thermus strains accumulated the (beta)-anomer only, whereas both anomers were found in R. marinus. 2-O-(beta)-mannosylglycerate and 2-O-(alpha)-mannosylglycerate were the major compatible solutes in R. marinus. The former was the predominant solute in cells grown in 2.0 and 4.0% NaCl-containing medium, while the latter was the predominant compatible solute at higher salinities. Glutamate, trehalose, and glucose were also present as minor components. The intracellular K(sup+) concentration, as determined by (sup39)K nuclear magnetic resonance spectroscopy, in R. marinus increased with salinity and was sufficient to balance the negative charges of the mannosylglycerate. In addition to 2-O-(beta)-mannosylglycerate, trehalose was a major compatible solute of "T. thermophilus." 2-O-(beta)-Mannosylglycerate was the main solute in medium containing 1.0 or 2.0% NaCl, while trehalose predominated in cells grown in medium supplemented with 3.0 or 4.0% NaCl. Glycine betaine, in lower concentrations, was also detected in two "T. thermophilus" strains. This is the first report of mannosylglycerate as a compatible solute in bacteria.     

Effects of osmotic stress on Methanococcus thermolithotrophicus: 13C-edited 1H-NMR studies of osmolyte turnover

In vivo NMR studies of the thermophilic archaeon Methanococcus thermolithotrophicus, with sodium formate as the substrate for methanogenesis, were used to monitor formate utilization, methane production, and osmolyte pool synthesis and turnover under different conditions. The rate of formate conversion to CO2 and H2 decreased for cells adapted to higher external NaCl, consistent with the slower doubling times for cells adapted to high external NaCl. However, when cells grown at one NaCl concentration were resuspended at a different NaCl, formate utilization rates increased. Production of methane from 13C pools varied little with external NaCl in nonstressed culture, but showed larger changes when cells were osmotically shocked. In the absence of osmotic stress, all three solutes used for osmotic balance in these cells, l-alpha-glutamate, beta-glutamate, and Nepsilon-acetyl-beta-lysine, had 13C turnover rates that increased with external NaCl concentration. Upon hyperosmotic stress, there was a net synthesis of alpha-glutamate (over a 30-min time-scale) with smaller amounts of beta-glutamate and little if any of the zwitterion Nepsilon-acetyl-beta-lysine. This is a marked contrast to adapted growth in high NaCl where Nepsilon-acetyl-beta-lysine is the dominant osmolyte. Hypoosmotic shock selectively enhanced beta-glutamate and Nepsilon-acetyl-beta-lysine turnover. These results are discussed in terms of the osmoadaptation strategies of M. thermolithotrophicus.     

Switching osmolyte strategies: response of Methanococcus thermolithotrophicus to changes in external NaCl

Methanococcus thermolithotrophicus, a thermophilic methanogenic archaeon, produces and accumulates beta-glutamate and L-alpha-glutamate as osmolytes when grown in media with <1 M NaCl. When the organism is adapted to grow in >1 M NaCl, a new zwitterionic solute, N(epsilon)-acetyl-beta-lysine, is synthesized and becomes the dominant osmolyte. Several techniques, including in vivo and in vitro NMR spectroscopy, HPLC analyses of ethanol extracts, and potassium atomic absorption, have been used to monitor the immediate response of M. thermolithotrophicus to osmotic stress. There is a temporal hierarchy in the response of intracellular osmolytes. Changes in intracellular K(+) occur within the first few minutes of altering the external NaCl. Upon hypoosmotic shock, K(+) is released from the cell; relatively small changes occur in the organic osmolyte pool on a longer time scale. Upon hyperosmotic shock, M. thermolithotrophicus immediately internalizes K(+), far more than would be needed stoichiometrically to balance the new salt concentration. This is followed by a decrease to a new K(+) concentration (over 10-15 min), at which point synthesis and accumulation of primarily L-alpha-glutamate occur. Once growth of the M. thermolithotrophicus culture begins, typically 30-100 min after the hyperosmotic shock, the intracellular levels of organic anions decrease and the zwitterion (N(epsilon)-acetyl-beta-lysine) begins to represent a larger fraction of the intracellular pool. The observation that N(epsilon)-acetyl-beta-lysine accumulation occurs in osmoadapted cells but not immediately after osmotic shock is consistent with the hypothesis that lysine 2,3-aminomutase, an enzyme involved in N(epsilon)-acetyl-beta-lysine synthesis, is either not present at high levels or has low activity in cells grown and adapted to lower NaCl. That lysine aminomutase specific activity is 8-fold lower in protein extracts from cells adapted to low NaCl compared to those adapted to 1.4 M NaCl supports this hypothesis.     

Ionic dependence of the extracellular ATP-induced permeabilization of transformed mouse fibroblasts: role of plasma membrane activities that regulate cell volume

Extracellular ATP rendered the plasma membrane of transformed mouse fibroblasts permeable to normally impermeant molecules. This permeability change was prevented by increasing the ionic strength of the isotonic medium with NaCl. Conversely, the cells exhibited increased sensitivity to ATP when the NaCl concentration was decreased below isotonicity, when the KCl concentration was increased above 5 mM while maintaining isotonicity, and when the pH of the medium was raised above 7.0. These conditions as well as the addition of ATP itself caused cell swelling. However, the effect of ATP was independent of cell volume and dependent upon the ionic strength and not the osmolarity of the medium since 1) addition of sucrose to isotonic medium did not prevent permeabilization although media made hypertonic with either sucrose or NaCl caused a decrease in cell volume; and 2) addition of sucrose or NaCl to hypotonic media caused a decrease in cell volume, but only NaCl addition decreased the response to ATP. Conditions that have been shown to inhibit plasma membrane proteins that play a reciprocal role in cell volume regulation had reciprocal effects on the permeabilization process, even though the effect of ATP was independent of cell volume. For example, inhibition of the Na+,K+-ATPase by ouabain increased sensitivity of cells to ATP while conditions which inhibit Na+,K+,Cl- -cotransporter activity, such as treatment of the cells with the diuretics furosemide or bumetanide or replacement of sodium chloride in the medium with sodium nitrate or thiocyanate, inhibited permeabilization. The furosemide concentration that inhibited permeabilization was greater than the concentration that inhibited Na+,K+,Cl- -cotransporter-mediated 86Rb+ (K+) uptake, suggesting that the effect of furosemide on the permeabilization process may not be specific for the Na+,K+,Cl- -cotransporter.     

Multiphasic osmotic adjustment in a euryhaline cyanobacterium

Abstract Transfer of Synechocystis PCC6714 from a freshwater medium to a saline medium caused the cells to shrink; rapid entry of NaCl resulted in a partial recovery of cellular volume within 2 min. Active extrusion of internal Na⁺ in exchange for extracellular K⁺ then occurred (within 20 min). Finally, the low- M _r carbohydrates sucrose and glucosylglycerol were accumulated and internal KC1 levels declined. In long-term growth experiments, the relative importance of sucrose as a component of the low- M _r organic solute fraction decreased and glucosylglycerol became the single most important intracellular solute. These observations demonstrate that several inorganic and organic solutes are involved in osmotic adjustment in this cyanobacterium, with sequential changes in the relative importance of each solute following transfer to a saline medium.     

Comparison of the function of L- and D-proline as compatible solute inEscherichia coli K-12 under high osmolarity

We compared the function of L- and D-proline as compatible solute inEscherichia coli K-12 cells under high osmolarity. Growth ofE. coli K-12 in a Davis minimal medium was inhibited at 0.5 M and 1 M NaCl, but it was recovered by the addition of L-proline. Glucose uptake was reduced with increase of external NaCl concentrations, but it was improved by the addition of L-proline. On the other hand, the addition of D-proline did not show the role of compatible solute although accumulated in cells. On the analysis ofE. coli proline transporter mutants, difference of the affinity of proline transporters for D-proline was observed at PutP and ProP. These results presumed that the functional disorder of D-proline as compatible solute was caused by its structural feature in cells.     

Sodium ion-stimulated alpha-[1-14C]aminoisobutyric acid uptake in alkalophilic Bacillus species.

Alkalophilic Bacillus no. 8-1 grows well in alkaline media containing 2.5 to 5% NaCl. The uptake of alpha-aminoisobutyric acid (AIB) into the cells is stimulated by the addition of NaCl (Na+) up to a concentration of 0.2 M, but other monovalent cations such as K+, Li+, or NH4+ cannot substitute for Na+. The kinetic studies reveal that, when the Na+ concentration increases from 0.02 to 0.2 M in alkaline medium, the Km for transport decreases, whereas Vmax remains almost constant. Competition studies indicate that glycine, L-alanine, L-serine, and AIB share common carriers for the transport of the compounds into cells. Other alkalophilic bacteria are also found to require Na+ for the uptake of AIB into the cells.     

13C NMR study of the interrelation between synthesis and uptake of compatible solutes in two moderately halophilic eubacteria. Bacterium Ba1 and Vibro costicola

The synthesis and uptake of intracellular organic osmolytes (compatible solutes) were studied with the aid of natural abundance 13C NMR spectroscopy in two unrelated, moderately halophilic eubacteria: Ba1 and Vibrio costicola. In minimal media containing 1 M NaCl, both microorganisms synthesized the cyclic amino acid, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (trivial name, ectoine) as the predominant compatible solute, provided that no glycine betaine was present in the growth medium. When, however, the minimal medium was supplemented with glycine betaine or the latter was a component of a complex medium, it was transported into the cells and the accumulating glycine betaine replaced the ectoine. In Ba1, grown in a defined medium containing glucose as the single carbon source, ectoine could only be detected if the NaCl concentration in the medium was higher than 0.6 M; the ectoine content increased with the external salt concentration. At NaCl concentrations below 0.6 M, alpha,alpha-trehalose was the major organic osmolyte. The concentration of ectoine reached its peak during the exponential phase and declined subsequently. In contrast, the accumulation of glycine betaine continued during the stationary phase. The results presented here indicate that, at least in the two microorganisms studied, ectoine plays an important role in haloadaptation.     

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.     

The use of metrizamide for stabilizing against convection in sedimentation equilibrium

In meniscus depletion sedimentation equilibrium experiments, the solute concentration gradient in much of the cell is insufficient to stabilize against thermal convection. Experiments with spectrin at 35 degrees C led to sufficient convection that, particularly at low concentrations of solute, no apparent redistribution occurred. At low angular velocity, the density gradient provided by moderate concentrations of NaCl or additional sucrose is inadequate to overcome the convective disturbances. We have found that the use of low concentrations of metrizamide can overcome these problems. The higher molecular weight and lower partial specific volume of metrizamide vis-a-vis sucrose allow a significant and stabilizing density gradient even at angular velocities below 8000 rpm.     

The role of osmotic effects in haloadaptation of Vibrio costicola

Growth rates of Vibrio costicola showed a broad optimum between 0.8 and 1.5 M-NaCl, and there was no growth above 3.3 M-NaCl in a peptone-based medium. The minimum requirement of 0.5 M-NaCl for growth in NaCl alone was reduced to 0.3 M-NaCl when the total solute concentration was raised to 0.5 to 1.0 M equivalent with sucrose or glycerol. Compared with equivalent NaCl concentrations, higher concentrations of sucrose were more inhibitory to growth, whereas glycerol had less effect. Increasing the medium NaCl concentration suddenly by 2- or 3-fold with either a constant starting, or final, salt concentration showed that, after the shift-up, the lag in growth, the rate of growth, and the inhibition of phospholipid synthesis depended both on the final NaCl concentration and the magnitude of the shift in salinity. The time-courses of phospholipid synthesis following a 2- or 3-fold shift-up in NaCl or sucrose media were very similar and exhibited a relative increase in phosphatidylglycerol synthesis over that of phosphatidylethanolamine. This 'switch-over' was not seen following shift-up in glycerol media when there was also a stimulation, rather than inhibition, of phospholipid synthesis. It is concluded that during phenotypic haloadaptation of V. costicola, osmotic effects play a significant part in the sensing of and response to raised external salinity.     

The response of plant mitochondria to media of high solute content

The state-3 rate of respiration of potato tuber mitochondria is inhibited by concentrations of KCl or NaCl above 125 mM, and by concentrations of sucrose, lactose, or maltose above 500 mM, but not at all by mannitol, glucose, glycine, or proline up to a concentration of 1500 mM in the medium. Mitochondria from cauliflower, beetroot, cucumber, rock melon, and watermelon behave very similarly to those from potato tuber. The variable response to different solutes proves that the reduction in respiration is not a simple function of the chemical potential of water in the medium. Disruption of potato mitochondria by ultrasonic vibration does not relieve the inhibition of succinate oxidation caused by KCl or sucrose. However, treatment with detergent abolishes completely the inhibition of respiration by sucrose. Inhibition of succinate dehydrogenase [Succinate:PMS, oxidoreductase (EC.1.3.99.1)] and malate dehydrogenase [L-Malate:NAD oxidoreductase (EC.1.1.1.37)] activities by sucrose is less than the inhibition of succinate- and malate-dependent oxygen uptake by the potato mitochondria. Limited substrate uptake and, alternatively, reduced electron flow as a consequence of a direct effect of solute on the mitochondrial membrane are considered as possible mechanisms of inhibition.     

Halotolerance in Methanosarcina spp.: Role of N(sup(epsilon))-Acetyl-(beta)-Lysine, (alpha)-Glutamate, Glycine Betaine, and K(sup+) as Compatible Solutes for Osmotic Adaptation

The methanogenic Archaea, like the Bacteria and Eucarya, possess several osmoregulatory strategies that enable them to adapt to osmotic changes in their environment. The physiological responses of Methanosarcina species to different osmotic pressures were studied in extracellular osmolalities ranging from 0.3 to 2.0 osmol/kg. Regardless of the isolation source, the maximum rate of growth for species from freshwater, sewage, and marine sources occurred in extracellular osmolalities between 0.62 and 1.0 osmol/kg and decreased to minimal detectable growth as the solute concentration approached 2.0 osmol/kg. The steady-state water-accessible volume of Methanosarcina thermophila showed a disproportionate decrease of 30% between 0.3 and 0.6 osmol/kg and then a linear decrease of 22% as the solute concentration in the media increased from 0.6 to 2.0 osmol/kg. The total intracellular K(sup+) ion concentration in M. thermophila increased from 0.12 to 0.5 mol/kg as the medium osmolality was raised from 0.3 to 1.0 osmol/kg and then remained above 0.4 mol/kg as extracellular osmolality was increased to 2.0 osmol/kg. Concurrent with K(sup+) accumulation, M. thermophila synthesized and accumulated (alpha)-glutamate as the predominant intracellular osmoprotectant in media containing up to 1.0 osmol of solute per kg. At medium osmolalities greater than 1.0 osmol/kg, the (alpha)-glutamate concentration leveled off and the zwitterionic (beta)-amino acid N(sup(epsilon))-acetyl-(beta)-lysine was synthesized, accumulating to an intracellular concentration exceeding 1.1 osmol/kg at an osmolality of 2.0 osmol/kg. When glycine betaine was added to culture medium, it caused partial repression of de novo (alpha)-glutamate and N(sup(epsilon))-acetyl-(beta)-lysine synthesis and was accumulated by the cell as the predominant compatible solute. The distribution and concentration of compatible solutes in eight strains representing five Methanosarcina spp. were similar to those found in M. thermophila grown in extracellular osmolalities of 0.3 and 2.0 osmol/kg. Results of this study demonstrate that the mechanism of halotolerance in Methanosarcina spp. involves the regulation of K(sup+), (alpha)-glutamate, N(sup(epsilon))-acetyl-(beta)-lysine, and glycine betaine accumulation in response to the osmotic effects of extracellular solute.     

The vesicle-to-micelle transition of phosphatidylcholine vesicles induced by nonionic detergents: effects of sodium chloride, sucrose and urea

The vesicle-to-micelle transition of egg phosphatidylcholine LUVs induced by octylglucoside was studied in buffers with 0-4 M sodium chloride, sucrose or urea. We used both light scattering and fluorescent probes to follow the lipid-detergent complexes in these buffers. The vesicle-to-micelle transition process was fundamentally the same in each solute. However, the detergent-to-lipid ratio required for micelle formation shifted in ways that depended on the aqueous solute. The partitioning of octylglucoside between the vesicles and the aqueous phase was primarily determined by the change in its critical micelle concentration (cmc) induced by each solute. Specifically, the cmc decreased in high salt and sucrose buffers but increased in high concentrations of urea. Cmc for two additional nonionic detergents, decyl- and dodecyl-maltoside, and three zwittergents (3-12, 3-14 and 3-16) were determined as a function of concentration for each of the solutes. In all cases NaCl and sucrose decreased the solubility of the detergents, whereas urea increased their solubilities. The effects clearly depended on acyl chain length in urea-containing solutions, but this dependence was less clear with increasing NaCl and sucrose concentrations. The contributions of these solutes to solubility and to interfacial interactions in the bilayers, pure and mixed micelles are considered.     

Effects of hyperosmolarity on ligand processing and receptor recycling in the hepatic galactosyl receptor system

Binding, endocytosis, and degradation of asialo-orosomucoid (ASOR) mediated by the galactosyl (Gal) receptor were examined in isolated rat hepatocytes in complete media supplemented with an osmolite. The specific binding of 125I-ASOR to cells at 4 degrees C was unaffected by up to 0.4 M sucrose or NaCl. Unlike sucrose or NaCl, mannitol stimulated 125I-ASOR binding at low concentrations but inhibited binding at higher concentrations. Continuous internalization at 37 degrees C, which requires receptor recycling, was completely blocked at 0.2 M sucrose or 0.15 M NaCl, corresponding in each case to a total osmolality of about 550 mmol/kg. This effect was reversed and endocytic function was restored by washing the cells, indicating that cell viability was unaffected. The rate of degradation of internalized 125I-ASOR was also inhibited by increasing sucrose concentrations. This inhibition is due to a block in the delivery of ligand to lysosomes and not an effect on degradation per se. In the presence of 0.2 M sucrose, the rate and extent of endocytosis of surface-bound 125I-ASOR were, respectively, 33.0 +/- 8.1% and 69.4 +/- 10.5% (n = 8) of the control without sucrose. Under these conditions, the dissociation of internalized receptor-ASOR complexes was completely inhibited. When sucrose was added, the effect on the endocytosis of surface-bound 125I-ASOR was virtually immediate. Previous studies showed that about 40% of the surface-bound 125I-ASOR which is internalized can return to the cell surface still bound to receptor (Weigel and Oka: J Biol Chem 259:1150, 1984). If 0.2 M sucrose was added after endocytosis occurred, 125I-ASOR still returned to the cell surface, although the rate and extent of return were inhibited by more than 50%. Interestingly, hyperosmolarity is the only treatment we have found which can reversibly inhibit, although only partially, the endocytosis of surface-bound 125I-ASOR.     

Composition, Variation, and Dynamics of Major Osmotic Solutes in Methanohalophilus Strain FDF1

Methanohalophilus strain FDF1, a member of the halophilic genus of methanogens, can grow over a range of external NaCl concentrations from 1.2 to 2.9 M and utilize methanol, trimethylamine, and dimethyl sulfide as substrates for methanogenesis. It produces the osmolytes glycine betaine, beta-glutamine, and N-acetyl-beta-lysine with increasing external NaCl, but the relative ratio of these zwitterions depends primarily on the methanogenic substrate and less on the external osmolarity. When the cells are grown on methanol in defined medium, accumulation of glycine betaine predominates over the other zwitterionic solutes. The cells also synthesized a carbohydrate which was not detected in cells grown on trimethylamine. This negatively charged compound, identified as alpha-glucosylglycerate from the C and H chemical shifts, does not act as an osmoregulatory solute in the salt range 1.4 to 2.7 M in this methanogen as evidenced by its invariant intracellular concentration. CH(3)OH-pulse/CH(3)OH-chase experiments were used to determine half-lifes for these organic solute pools in the cells. l-alpha-Glutamate showed a rapid loss of heavy isotope, indicating that l-alpha-glutamate functions as a biosynthetic intermediate in these cells. Measurable turnover rates for both beta-glutamine, which acts as an osmolyte, and alpha-glucosylglycerate suggest that they function as metabolic intermediates as well. Molecules which function solely as osmolytes (glycine betaine and N-acetyl-beta-lysine) showed a slower turnover consistent with their roles as osmotic solutes in Methanohalophilus strain FDF1.