Glucose-regulated membrane properties of untransformed and virus-transformed BHK21 cells.

BHK21 fibroblasts transformed by hamster sarcoma virus have a higher rate of uptake of hexoses than their untransformed counterparts, and therefore rapidly exhaust glucose from the culture medium. The effects of culturing normal and transformed BHK cells, both in limiting and in excess glucose, on several membrane properties related to malignant transformation have been studied. The increase in the rate of hexose uptake in transformed cells is partially but not entirely dependent on extracellular glucose concentration. Two transformation-increased membrane proteins of molecular weights 95 000 and 78 000 are shown to be regulated by extracellular glucose concentration in both normal and transformed cells. The loss of LETS-protein, the high density of intramembranous particles, the increase in the amount of a 177K integral plasma membrane protein and the increase in the amount of high molecular weight surface glycopeptides in transformed cells, are not related to glucose depletion of the medium. Beside LETS, another iodinated protein, of molecular weight 160 000, is decreased in transformed cells. The exposure of this protein increased in both normal and transformed cells when arrested in G1 by asparagine deprivation.     

On the susceptibility of human platelets to aggregation by concanavalin A and the effect of this lectin on their response to ADP

Concanavalin A aggregated gel-filtered platetes in 0.9% NaCl solution signifying cross-bridging by the lectin. Aggregation of these platelets by concanavalin A was temperature dependent; it did not occur at 0–4 °C unless the platelets were previously trypsinized. The level of aggregation of trypsinized platelets by concanavalin A at 0–4°C was similar to that of untreated platelets at 37°C. It is suggested that trypsin facilitates platelet aggregation by concanavalin A at 0–4°C by causing a configurational change in membrane glycoproteins which orientates concanavalin A receptor sites into positions that favour lectin cross-bridging. Concanavalin A failed to aggregate platelets in plasma. Radioisotope studies showed that the amount of [³H]concanavalin A which combined with platelets in plasma was extremely low compared with gel-filtered platelets in saline. The aggregation of Ehrlich ascites cells by concanavalin A was considerably reduced when platelet-free plasma was added to the medium suggesting that it was due to the presence of concanavalin A-reactive components in the plasma.Concanavalin A inhibited the ADP-induced aggregation of platelets suspended in plasma or in a salts solution supplemented with calcium and fibrinogen, although the inhibitory effect was more conspicuous in the latter case. The results suggests that concanavalin A produces its inhibitory effect on ADP-induced platelet aggregation by interacting with membrane glycoproteins, and this further suggests their involvement in aggregation.     

Regulation of the plasma membrane during exposure to low temperatures in suspension-cultured cells from a cryophyte (Chorispora bungeana)

Summary. As the outermost boundary of the cell, the plasma membrane plays an important role in determining the stress resistance of organisms. To test this concept in a cryophyte, we analyzed alterations of several components in plasma membranes isolated from suspension-cultured cells of Chorispora bungeana Fisch. & C.A. Mey in response to treatment at 0 and −4 °C for 192 h. When compared with the controls growing at 25 °C, both the membrane permeability and fluidity showed recovery after the initial impairment. Linolenic acid and membrane lipid unsaturation increased by about 0.8-fold following cold treatments, although the kinetics of the increase varied with the temperatures examined. During the treatments, the plasma membrane H⁺-ATPase (EC 3.6.1.3) activity increased by 78.06% at 0 °C and 100.47% at −4 °C. However, the plasma membrane NADH oxidase (EC 1.6.99.3) activity only decreased when exposed to a lower temperature (−4 °C), and remained at 63.93% after being treated for 192 h. After the treatments, the physical properties of the plasma membranes of suspension-cultured cells, especially the −4 °C treated cells, were similar to those in the wild plants. These findings indicate that the specific mechanism of cold resistance of C. bungeana is tightly linked with the rapid and flexible regulation of membrane lipids and membrane-associated enzymes, which ensure the structural and functional integrity of the plasma membrane that is essential for withstanding low temperature. Correspondence: Lizhe An, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China.     

Variation of frequency spectrum of the erythrocyte flickering caused by aging,osmolarity, temperature and pathological changes

Frequency spectra of the surface undulations (flickering) of erythrocyte plasma membranes are measured by direct spectral analysis of the intensity fluctuations of the light passing the cells in a phase contrast microscope. Spectra are taken as a function (1) of the temperature (2) of the viscosity and osmolarity of the outer medium (3) of the aging of cells and (4) of pathological transformations. The spectra are approximately superpositions of two Lorentzian lines. At large frequencies,f, the spectra follow f^?2. This behaviour can be interpreted in terms of cell thickness fluctuations caused by thermally excited membrane undulations provided the range of wavelengths is small. The undulations are determined by the membrane curvature elasticity while the lateral tension is negligibly small for cells of discoid shape. The technique presented allows accurate measurements of relative curvature (bending) elastic constants. The spectra of freshly drawn cells are remarkably reproducible. Aging of the cells in the medium leads to an increase in the curvature elastic constant. A decrease in osmolarity causes a reduction in the intensity and line width of the spectra and the flickering vanishes if the cell approaches a spherical shape. The effect of temperature between 10 and 40°C is astonishingly small with the exception of a sudden increase in the amplitude with increasing temperature at 35°C. The flicker spectra of a large fraction of the cells from patients suffering from cronical alcoholism exhibit a reduced line width or an increase in the curvature elastic constant.     

Effects of semen preservation on boar spermatozoa head membranes

Head plasma membranes were isolated from the sperm-rich fraction of boar semen and from sperm-rich semen that had been subjected to three commercial preservation processes: Ex tended for fresh insemination (extended), prepared for freezing but not frozen (cooled), and stored frozen for 3-5 weeks (frozen-thawed). Fluorescence polarization was used to determine fluidity of the membranes of all samples for 160 min at 25°C and also for membranes from the sperm-rich and extended semen during cooling and reheating (25 to 5 to 40°C, 0.4°C/min). Head plasma membranes from extended semen were initially more fluid than from other sources (P < 0.05). Fluidity of head membranes from all sources decreased at 25°C, but the rate of decrease was significantly lower for membranes from cooled and lower again for membranes from frozen-thawed semen. Cooling to 5°C reduced the rate of fluidity change for plasma membranes from the spernvrich fraction, while heating over 30°C caused a signifi cantly greater decrease. The presence of Ca⁺⁺ (10 mM) lowered the fluidity of the head plasma membranes from sperm-rich and extended semen over time at 25°C but did not affect the membranes from the cooled or frozen-thawed semen. The change in head plasma membrane fluidity at 25°C may reflect the dynamic nature of spermatozoa membranes prior to fertilization. Extenders, preservation processes and temperature changes have a strong influence on head plasma membrane fluidity and therefore the molecular organization of this membrane.     

The eggs of the freshwater fish Epiplatys dageti have tight plasma membranes without intramembranous particles

Summary The plasma membrane of fish (Epiplatys dageti) eggs, which are capable of developing in salt-free water despite intracellular osmolarity corresponding to a pressure gradient of 7 to 8 atmospheres, is almost devoid of intramembrane particles. The specific membrane resistance is at least 3 orders of magnitude larger than that of nerve or muscle cells of different species indicating that the membrane is tight. These findings support the view that intramembranous particles are involved in transmembrane transport of ions, and indicate that the ionic concentration gradient in this cell is maintained by a tight membrane rather than by active transport.     

Effect of extraction temperature on the diffusion coefficient of polysaccharides from Spirulina and the optimal separation method

The extraction temperature had a significant impact on the concentration of polysaccharides derived from solid-liquid extraction of Spirulina. The polysaccharide concentration was significantly higher when the extraction was performed at 90°C than when it was performed at 80, 70, and 50°C. This result is related to the diffusion coefficients of the polysaccharides, which increased from 1.07 × 10^?12 at 50°C to 3.02 × 10^?12 m²/sec at 90°C. Using the Arrhenius equation, the pre-exponential factor (D ₀ ) and the activation energy (E _a ) for Spirulina polysaccharide extraction were calculated as 7.958 × 10^?9 m²/sec and 24.0 kJ/mol, respectively. Among the methods used for the separation of Spirulina polysaccharides, cetyltrimethylammonium bromide (CTAB, method I) and organic solvent (ethanol, in methods II and III) provided similar yields of polysaccharides. However, the separation of polysaccharides using an ultrafiltration (UF) process (method III) and ethanol precipitation was superior to separation via CTAB or vacuum rotary evaporation (method II). The use of a membrane with a molecular weight cut-off (MWCO) of 30 kDa and an area of 0.01 m² at a feed pressure of 103 kPa with a mean permeate flux of 39.3 L/m²/h and a retention rate of 95% was optimal for the UF process. The addition of two volumes (v/v) of ethanol, which gave a total polysaccharide content of approximately 4% dry weight, was found to be most suitable for polysaccharide precipitation. The results of a Sepharose 6B column separation showed that the molecular weights of the polysaccharides in fractions I and II were 212 and 12.6 kDa, respectively.     

Corticotropin stimulates cyclic nucleotide independent protein kinase activity of intact adrenocortical cells

Intact adrenocortical cells possess cyclic nucleotide-independent protein kinase activity which is capable of phosphorylating endogenous proteins and casein when incubated in the presence of [γ-³²P]ATP. The cyclic nucleotide-independent enzyme was dependent on cell number and temperature and had an apparent K_m for ATP of 6.5 × 10^?5 M and a V_max of 12.5 pmol/3 min/2 × 10⁵ cells at 37°C. Phosphorylation of endogenous proteins by this kinase was increased by treatment of intact cells with corticotropin (2.2 nM) for 24 h. In control cells, two endogenous proteins of apparent molecular weights of 39,000 and 76,000 were phosphorylated. In corticotropin-treated cells, another protein of apparent molecular weight of 87,000 was also phosphorylated. Thus, this protein kinase activity, which appears to be located on the plasma membrane, may be involved in mediating longer term actions of corticotropin on the adrenal cortex.     

A Freeze-Fracture and Concanavalin A-Binding Study of the Membrane of Cleaving Xenopus Embryos

The freeze-fracture appearance and concanavalin A-binding capacity of the plasma membrane of cells of the cleaving Xenopus embryo have been examined up to the 16-cell stage. It was found that membrane on the outer surface of the embryo, which faces the vitelline membrane and is remote from cleavage furrows, and membrane in the shallow regions of the furrow possessed a high population of intramembranous particles on the PF-face (1171 per μm²). The EF-face of these membranes showed a lower particle population (245 per μm²). By contrast, membrane deep in the furrow and bounding the blastocoel did not display a face with high particle numbers. Both faces of this membrane, which is newly exposed as the furrow grows, were relatively poorly supplied with particles (93 per μm²). Therefore it appears that, in this tissue, newly added membrane possesses fewer intramembranous particles than the pre-existing membrane. Concanavalin A, as detected cytochemically using peroxidase and haemocyanin techniques, bound extensively to both particle-rich and particle-poor membrane. Thus there was no correlation between intramembranous particle frequency and degree of concanavalin A binding.     

Temperature-Induced Inactivation of Cytoplasmic Biogel Osmosensing Properties is Associated with Suppression of Regulatory Volume Decrease in A549 Cells

Upstream intermediates of intracellular signaling involved in cell volume regulation remain poorly explored. Recently, we demonstrated that osmolarity-induced volume changes in permeabilized cells were several-fold higher than those observed with intact cells, indicating the osmosensing properties of cytoplasmic gel. To further examine the role of cytoplasmic biogel in cell volume regulation, we compared the action of short-term heat treatment on volume changes in intact and permeabilized A549 cells. Pretreatment of A549 cells at 48 °C suppressed swelling triggered by dissipation of Donnan’s equilibrium as well as by hyposmotic medium. Significantly, heat treatment completely abolished the action of hyposomotic medium on volume changes in permeabilized cells, showing that temperature elevation suppresses osmosensing properties via its effect on biogel rather than on plasma membrane water permeability. Identical heat treatment blocked the regulatory volume decrease (RVD) as well as the increment of Ba²⁺-sensitive K⁺-channel activity seen in control cells exposed to hyposmotic swelling. Unlike swelling, hyperosmotic shrinkage was decreased by twofold in cells subjected to 10-min preincubation at 50 °C. Our results disclose that osmosensing by cytoplasmic gel is a key event in the RVD triggered by hypotonic swelling. The role of biogel and plasma membrane in intracellular signaling triggered by hyperosmotic shrinkage should be further investigated.     

Rearrangement of intramembranous particles and fusion promoted in chicken erythrocytes by intracellular Ca2+

Ca²⁺ was introduced into fresh and ATP-depleted chicken erythrocytes through the aid of the ionophore A-23187.Intracellular Ca²⁺ (10–40 mM) induced fusion in ATP-depleted cells after 30–60 min incubation at 37°C, but not in fresh cells. Fresh cells underwent a higher degree of haemolysis than ATP-depleted cells after accumulation of Ca²⁺. Uptake of Ca²⁺ was the same in these two systems.Intracellular Ca²⁺ induced rearrangement of intramembranous particles, as revealed by freeze-etching studies. The intramembranous particles in the protoplasmic face of fractured membranes obtained from fresh cells incubated with 1 mM of Ca²⁺ were more scattered and their density was lower than in control cells. Incubation with higher concentrations of Ca²⁺ (10–40 mM) induced transient changes in the intramembranous particles' density with the appearance of protrusions and depressions on the protoplasmic and exoplasmic faces of the fractured membranes, respectively. These effects were reversible upon removal of Ca²⁺ by washing the cells with ethyleneglycol $\text{bis}\text{(α-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid; rearrangement of intramembranous particles was less evident after accumulation of Ca²⁺ in ATP-depleted cells, whose fractured membranes did not contain any protrusions or depressions.Transferring Ca²⁺-loaded cells to the cold caused the formation of large smooth areas devoid of intramembranous particles in the protoplasmic face of the fractured membranes.Cells containing Ca²⁺ appeared spherical, and removal of Ca²⁺ restored the normal oval shape of chicken erythrocytes.     

Freeze-fracture planes of methanogen membranes correlate with the content of tetraether lipids.

Methanospirillum hungatei GP1 contained 50% of its ether core lipids (polar lipids less head groups) as tetraether lipids, and its plasma membrane failed to fracture along its hydrophobic domain during freeze-etching. The membrane of Methanosaeta ("Methanothrix") concilii did not contain tetraether lipids and easily fractured to reveal typical intramembranous particles. Methanococcus jannaschii grown at 50 degrees C contained 20% tetraether core lipids, which increased to 45% when cells were grown at 70 degrees C. The frequency of membrane fracture was reduced as the membrane-spanning tetraether lipids approached 45%. As the tetraether lipid content increased, and while fracture was still possible, the particle density in the membrane increased; these added particles could be tetraether lipid complexes torn from the opposing membrane face. The diether membrane (no tetraether lipid) of Methanococcus voltae easily fractured, and the intramembranous particle density was low. Protein-free liposomes containing tetraether core lipids (ca. 45%) also did not fracture, whereas those made up exclusively of diether lipids did split, indicating that tetraether lipids add considerable vertical stability to the membrane. At tetraether lipid concentrations below 45%, liposome bilayers fractured to reveal small intramembranous particles which we interpret to be tetraether lipid complexes.     

Replica-moulded polydimethylsiloxane culture vessel lids attenuate osmotic drift in long-term cell cultures

An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity over time. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO₂ incubator at 37°C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/day. Depending on the lid membrane thickness, pH drifts at ambient CO₂ levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60°C yielded a 10-fold reduced water vapour flux of 1.75 g/day/dm² through PDMS lids as compared with 18.69 g/day/dm² with conventional Petri dishes. Using such PDMS lids, about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these, the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Supplementary material pertaining to this article is available on the Journal of Biosciences Website at     

Effects of amphotericin B and its methyl ester on plasma membranes of Candida albicans and erythrocytes as examined by freeze-fracture electron microscopy

Freeze-fracture electron microscopy of the plasma membranes of Candida albicans yeast cells and red blood cells treated with amphotericin methyl ester and amphotericin B showed that amphotericin B (50 micrograms ml-1) caused extreme aggregation of intramembranous particles on the protoplasmic fracture face of the C. albicans membrane, and a marked reduction of the density of intramembranous particles. On the other hand, the rearrangement of intramembranous particles induced by amphotericin methyl ester (50 micrograms ml-1) produced elevations of the particle-free membrane domains toward the outside of the cells, so that the particles were aggregated in linear furrows surrounding these elevations on the protoplasmic fracture face, and the corresponding ridges on the exoplasmic fracture face. The density of intramembranous particles was greatly reduced on the protoplasmic fracture face. Both polyenes produced only small changes in the erythrocyte membranes at the same concentration. These results suggest that amphotericin methyl ester affects the ergosterol-containing membranes more than amphotericin B, and that ergosterol has a higher sensitivity for these two polyene antibiotics than cholesterol.     

Spin label studies on rat liver and heart plasma membranes: Effects of temperature,calcium, and lanthanum on membrane fluidity

The structures of rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(1 2,3). The polarity-corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(1 2,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity-corrected order parameters indicated that lipid phase separations occur in liver (between 19° and 28°C) and heart (between 21° and 32°C) plasma membranes. The possibility that a wide variety of membrane-associated functions may be influenced by these thermotropic phase separations is considered. Addition of 3.9 mM CaCl₂ to I(1 2,3)-labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37°C. Similarly, titrating I(1 2,3)-labeled heart plasma membranes with either CaCl₂ or LaCl₃ decreased the lipid fluidity at 37°C, although the magnitude of the La³⁺ effect was larger and occurred at lower concentrations than that induced by Ca²⁺; addition of 0.2 mM La³⁺ or 3.2 mM Ca²⁺ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe–probe interactions. Ca²⁺ and La³⁺ at these concentrations decrease the activities of such plasma membrane enzymes as Na⁺, K⁺-ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation-mediated decreases in the lipid fluidity.     

Mitochondrial function in Antarctic notothenioid fishes that differ in the expression of oxygen-binding proteins

State III respiration rates were measured in mitochondria isolated from hearts of Antarctic notothenioid fishes that differ in the expression of hemoglobin (Hb) and myoglobin (Mb). Respiration rates were measured at temperatures between 2 and 40°C in Gobionotothen gibberifrons (+Hb/+Mb), Chaenocephalus aceratus (–Hb/–Mb) and Chionodraco rastrospinosus (–Hb/+Mb). Blood osmolarity was measured in all three species and physiological buffers prepared for isolating mitochondria and measuring respiration rates. Respiration rates were higher in mitochondria from G. gibberifrons compared to those from C. aceratus at 2°C, but were similar among all species at temperatures between 10 and 26°C. Respiration rates were significantly lower in icefishes at 35 and 40°C compared to G. gibberifrons. The respiratory control ratio of isolated mitochondria was lower in C. aceratus compared to G. gibberifrons at all temperatures below 35°C. At 35 and 40°C, mitochondria were uncoupled in all species. The Arrhenius break temperature of state III respiration was similar among all three species (30.5 ± 0.9°C) and higher than values previously reported for Antarctic notothenioids, likely due to the higher osmolarity of buffers used in this study. These results suggest that differences in mitochondrial structure, correlated with the expression of oxygen-binding proteins, minimally impact mitochondrial function.     

Distribution of intramembranous particles and filipin-sterol complexes in the spermatid and spermatozoon of Culex quinquefasciatus (Culicidae).

A freeze-fracture study was carried out on spermatid and spermatozoon of the mosquito Culex quinquefasciatus. In the spermatid plasma membrane few and randomly distributed intramembranous particles were observed. In the spermatozoon the density of intramembranous particles was higher on the P- than on the E-fracture face of the plasma membrane. Two populations of particles were observed. Large particles (about 15 nm in diameter) are regularly arranged in double rows as a zipper-line, longitudinally oriented in relation to the main cell axis. These strands of particles were observed in the posterior head region, mainly associated with the E-fracture face. Filipin was used to analyse the presence and distribution of cholesterol in thin sections and freeze-fracture replicas. Filipin-sterol complexes were not homogeneously distributed throughout the spermatozoon plasma membrane. They were more abundant on the P-fracture face of the membrane lining the nuclear region. The results obtained show that Culex spermatozoon differs from those of other species in that its plasma membrane exhibits only a membrane domain, the zipper-line, localized in the postacrosomal region.     

The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.     

Tritrichomonas foetus: Fine Structure of Freeze-Fractured Membranes1

ABSTRACT. Freeze-fracture techniques reveal differences in fine structure between the anterior three flagella of Tritrichomonas foetus and its recurrent flagellum. The anterior flagella have rosettes of 9–12 intramembranous particles on both the P and E faces. The recurrent flagellum lacks rosettes but has ribbon-like arrays of particles along the length of the flagellum, which may be involved in the flagellum's attachment to the cell body. This flagellum is attached to the membrane of the cell body along a distinct groove that contains few discernible particles. Some large intramembranous particles are visible on the P face of the cell body membrane at the point where the flagellum emerges from the cell body. The randomly distributed particles on the P and E faces of the plasma membrane have a particle density of 919/μm² and 468/μm² respectively, and there are areas on both faces that are devoid of particles. Freeze-fracture techniques also reveal numerous fenestrations in the membrane of the Golgi complex and about 24 pores per μm² in the nuclear. membrane.     

Effects of temperature on growth and metabolism in juvenile turbot

The effects of constant temperatures on growth, food efficiency, and physiological status were studied in four different batches of juvenile turbot. The growth responses were studied in three experiments lasting 70–85 days under 8–20° C thermal conditions. There was a positive correlation between growth and temperature from 8 to 17° C and a plateau was observed from 17 to 20° C. In fish fed to satiety, specific growth rate was positively correlated to the food intake, which was double at 20° C, compared with 8° C. Minor changes were observed in food efficiency. Body fat deposition decreased as temperature increased (25% lower at 20° C, compared with 8° C). Apparent food conversion, PER (protein efficiency ratio) and PUC (protein utilization coefficient) ranges were 0.8–0.9, 2.1–2.3 and 33–38% respectively. In 70–300 g fish, routine MO₂ increased (2.5–6.5 μmol O₂ h^?1 g bw^?1) with temperature up to 20° C, while larger turbot (500–600 g) appeared relatively thermo-independent, with a lower oxygen consumption (1.5 ìmol h^?1 g^?1). The average daily total ammonia nitrogen (TAN) and urea-N excretion per fish biomass was positively related to temperature. TAN was 30% lower at 8° C, compared with 20° C. Ingested nitrogen was mainly excreted under the final form of TAN, urea-N representing 26% of the total amount. A post-prandial peak in TAN and a delayed peak in urea-N nitrogen were observed. The hydromineral status [osmolarity, sodium, chloride and potassium blood plasma, gill (Na⁺-K⁺)-ATPase activity] of turbot was not affected by progressive changes in temperature during the acclimation period. Juvenile turbots show remarkable homeostatic capacities and so they have a relatively thermo-independent physiology within the range of temperature studied.