The isolation of skeletal muscle plasma membranes from rainbow trout (Salmo gairdneri)

• 1.1. Plasma membranes were isolated from caudal flank skeletal musculature of rainbow trout by discontinuous sucrose gradient centrifugation.
• 2.2. Na⁺−K⁺-ATPase was enriched 8-fold and 5′-nucleotidase activities 4-fold in a fraction isolated at the 8–25% sucrose interface.
• 3.3. A cholesterol: phospholipid ratio of 0.37 in the plasma membrane fraction was 85% greater than that observed in adjacent subcellular fractions.
• 4.4. Electron microscopy provided morphological confirmation of enrichment and integrity of skeletal muscle plasma membranes at the 8–25% sucrose interface.

     

The influence of calcium and magnesium on sea bass (Dicentrarchus labrax) intestinal brush border membrane purification and activity

• 1.1. The activity of brush border enzymes (alkaline phosphatase, maltase, sucrase, trehalase, leucine amino peptidase) was higher in purified membranes prepared with calcium. The contamination of these membranes with basolateral membranes was also lower (1.27 for Na-K-ATPase activity ratio).
• 2.2. The extraction of brush border lipids was carried out according to Folch adapted method. Two dimensional thin layer chromatography was used to separate the phospholipidic fractions. Fatty acids of phospholipids were analysed using gas chromatography after acid transmethylation (column SP 2330).
• 3.3. Phospholipids are composed of phosphatidylcholine (PC: 33%), phosphatidylethanolamine (PE: 30%), sphingomyeline (SM: 21%), phosphatidylserine (PS: 14%) and phosphatidylinositol (PI: 2%). 4. PC, PE and PS are characterized by high levels of unsaturated fatty acids (monounsaturated MUFA: 21.5% and polyunsaturated PUFA: 34.9%). The most abundant PUFA belong to the (n-3) family [18:3 (n-3), 20:5 (n-3) and 22:6 (n-3)].
• 4.5. Fatty acids from sphingomyelin of purified membranes have low proportions of PUFA (13.5%) but higher proportions of MUFA (39.5%).
• 5.6. No specific differences were found between calcium and magnesium prepared membranes.
• 6.7. The low content in LPC and the absence of LPE confirmed the absence of major structural lipids transformation during the membrane purification with calcium or magnesium.
• 7.8. Glycine transport was measured during 10 sec at different temperatures using the rapid filtration technique. Glycine transport was higher with Na⁺ than with K⁺. In the presence of Na⁺, this transport increases with temperature.
• 8.9. Arrhenius curves were mono phasic without obvious breakpoint and indicated no phase transition in the lipid bilayer.
• 9.10. A significant Na⁺ dependent glycine transport has been characterized at low temperatures (0°C) which suggests a possible role of membrane polyunsaturated fatty acids in the control of glycine transport.

     

Sarcolemmal membranes from hamster and chicken skeletal muscle: Isolation and chemical composition

• 1.1. Plasma membranes were obtained from hamster (Mesocricetus auratus Wateth.) and chicken (Callus gallus L.). Skeletal muscle was isolated by muscle homogenization, protein extraction by inorganic salt solutions (0.4 M LiBr and 0.6 M KCl) and differential centrifugation. After purification on a discontinuous sucrose gradient, several fractions were obtained. The upper fraction (20% sucrose, w/w) yielded in the form of vesicles by electron microscope examination.
• 2.2. (Na⁺ + K⁺ )-ATPase and 5'-nucleotidase as plasma membrane markers were found to be concentrated in the upper fraction. Practically no succinate dehydrogenase activity was detected.
• 3.3. By means of polyacrylamide gel electrophoresis it was possible to separate 7–8 protein bands the molecular weights of which range from 26,000 to 200,000 daltons; and 4 bands for glycoproteins.
• 4.4. The ratio lipid: protein and the molar ratio cholesterol :phospholipid were found to be 0.93–0.94 and 0.25–0.38, respectively.
• 5.5. Glucose, mannose, galactose and fucose, hexosamines and sialic acids were determined in these preparations of membranes.

     

Small membrane-associated gtp-binding proteins of catecholamine-secreting cells

• 1.1. Four GTP-binding proteins (23–27 kDa) were identified in membranes from PC12 cells by [α³²P]GTP binding to nitrocellulose blots of SDS-polyacrylamide gels.
• 2.2. The GTP-binding proteins remained associated with membranes during stimulation of intact cells by K⁺-depolarization or even after addition of C²⁺to digitonin-permeabilized cells.
• 3.3. By two-dimensional gel electrophoresis, six GTP-binding proteins were resolved and based on their mobility, their phosphorylation state appeared independent of Ca²⁺.
• 4.4. Fractionation of PC12 membranes showed that these GTP-binding proteins were broadly distributed in post-nuclear membranes with the plasma membranes containing the highest specific GTP-binding activity.
• 5.5. Membrane fractions from bovine adrenal medulla contain similar GTP-binding proteins with GTP-binding intensity also being highest in the plasma membrane.
• 6.6. The GTP-binding proteins could be concentrated in the detergent-rich fraction upon Triton X-114 phase separation.

     

Differential post-receptor responses of adenylate cyclase in white and brown adipose tissue membranes of rats fed high-energy diets

• 1.1. Adenylate cyclase activity was determined in membranes of white and brown adipose tissue (WAT and BAT, respectively) from rats fed a high-energy diet (EXP group) vs those fed a nutritionally balanced one (CON group).
• 2.2. The isoproterenol- and guanine nucleotide-induced adenylate cyclase activity in WAT membranes of EXP rats was lower than that in CON rats.
• 3.3. Relative adenylate cyclase activity in like treated BAT membranes was higher in EXP than in CON rats.
• 4.4. It is concluded that feeding high-energy diets to rats induces similar post-receptor modifications of adenylate cyclase as found in genetic obese rodents.

     

Comparative aspects of plasma membrane-bound enzymes in frog and rat liver

• 1.1. Plasma membranes have been isolated from frog (Rana esculenta) liver.
• 2.2. The average yield was 0.194 mg protein/g wet liver.
• 3.3. The activities of plasma membrane-bound enzymes (Na⁺-K⁺-ATPase and 5'-nucleotidase as well as of (Mg²⁺)-ATPase have been determined in the liver homogenate and in isolated plasma membranes.
• 4.4. (Na⁺-K⁺-ATPase, 5'-nucleotidase and (Mg²⁺)-ATPase activities of frog liver are compared with the same enzymatic activities observed in rat liver.

     

Na+-independent sugar uptake by rat intestinal and renal brush border and basolateral membrane vesicles

• 1.1. Uptake of [¹⁴C]-labelled d-glucose, l-arabinose and d-fructose by intestinal and renal brush border and basolateral membrane vesicles was studied in the absence of Na⁺ .
• 2.2. The Na⁺-independent d-glucose transport system in these membrane vesicles was saturable, sensitive to phloretin, stereospecific and accessible only to d-glucose and d-galactose.
• 3.3. Na⁺-independent l-arabinose transport was not saturable even when its concentration was raised to 300 mM and it was insensitive to phloretin.
• 4.4. Na⁺-independent d-fructose transport demonstrated saturation kinetics with only renal brush border membrane vesicles, but it was not inhibited by either phloretin or phlorizin.
• 5.5. These studies indicated that the Na⁺-independent carrier-mediated d-glucose/d-galactose transport system of intestinal and renal brush border and basolateral membranes is clearly not shared by other monosaccharides.

     

Effect of caffeine,theophylline and nicotine on d-glucose and folate transport in rat jejunal brush border membrane vesicles

• 1.1. Nicotine at 10 mM, but not caffeine or theophylline, reduced by 20% the overshoot of the Na⁺-dependent d-glucose transport in ratjejunal brush border membrane vesicles.
• 2.2. Since nicotine did not affect the transport of Na⁺, its inhibition on Na⁺-dependent d-glucose transport must be due to a direct effect upon the d-glucose transport system.
• 3.3. Folate transport in these membrane vesicles was found to a be a free diffusion process at pH 7.4.
• 4.4. Neither caffeine, theophylline nor nicotine has any effect on folate transport.

     

Effects of water stress on hemolymph volume,osmotic potential and chemical composition in Megetra cancellata

• 1.1. Rates of water loss in Megetra cancellata were very high compared to those reported for other xeric arthropods.
• 2.2. Hemolymph weight in hydrated animals was 43.0% of the total body weight while it was 24.7% in desiccated animals that had lost 16.1% of their body weight as water.
• 3.3. Hemolymph osmotic potential increased from 417 to 447 mOsm/kg in desiccated beetles, but osmotic regulation was evident.
• 4.4. Total hemolymph protein mass and concentration decreased in desiccated beetles while amino acid concentrations remained constant (at about 70 mM).
• 5.5. Na⁺ and −PO₄ concentrations increased in desiccated beetles.
• 6.6. Cl⁻ and K⁺ concentrations in desiccated beetles were equal to those in undesiccated beetles.

     

Lysis of resealed erythrocyte ghosts by photoactivated tetrapyrroles: Estimation of photolesion dimensions

• 1.1. Resealed erythrocyte ghosts containing Na⁺ and glucose-6-P (G6P) as markers of membrane integrity were used as a model system for probing the damaging effects of photoactivated tetrapyrroles on cell membranes.
• 2.2. Continuous blue-light irradiation of bilirubin (BR)-sensitized and protoporphyrin (PP)-sensitized ghosts made them progressively more permeable to Na⁺ the cation emerging well ahead of G6P.
• 3.3. G6P efflux occurred abruptly after a lag period and resembled an all-or-none process.
• 4.4. These and other results suggest that a relatively subtle structural modification (possibly in some crucial protein(s) is sufficient for Na⁺ release, whereas gross disruption of the bilayer (probably by free-radical lipid peroxidation) is necessary for G6P release.
• 5.5. The dimensions of the G6P-releasing photolesions were estimated by density floatation centrifugation, using saccharides of increasing molecular size. Both BR and PP produced pores > 11 Å but <42 Å in diam, which is considerably smaller than the size range estimated in hypotonically lysed ghosts.

     

Blood volume recovery in hemorrhaged japanese quail

• 1.1. Blood volume and plasma biochemical changes and feed and water consumption in response to a hemorrhage by phlebotomy of 30% of the calculated total blood volume with and without replacement of blood volume with physiological saline were determined in juvenile male Coturnix coturnix japonica.
• 2.2. Plasma protein and osmolality decreased rapidly posthemorrhage and did not recover by 72 hr posthemorrhage.
• 3.3. Plasma glucose, Na⁺ and K⁺ increased within Ihr postphlebotomy. Plasma Na⁺ returned to nonphlebotomized levels within 6 hr postphlebotomy.
• 4.4. Saline replacement of blood volume resulted in hypervolemia within 3–5 min postphlebotomy.
• 5.5. Phlebotomized quail receiving no saline recovered blood volume to 0 hr (nonphlebotomized) levels within l hr postphlebotomy.

     

Some observations on the behaviour of the sodium efflux in barnacle muscle fibres towards lithium ions

• 1.1. The behaviour of the Na efflux towards Li⁺ was studied using single barnacle muscle fibres as a preparation.
• 2.2. It is found that the Na efflux into Li⁺-ASW (artificial seawater) is reduced and that this effect is not fully reversed by returning back to Na⁺-ASW.
• 3.3. Preinjection of 100 mM-EGTA reduces the magnitude of the fall of the Na efflux into Li⁺-ASW.
• 4.4(a). The remaining Na efflux into Li⁺-ASW is further reduced by external application of 10⁻⁴ M-ouabain. (b) The remaining Na efflux in ouabain-poisoned fibres is reduced by replacing Na_e by Li⁺. However, some fibres show a rise rather than a fall.
• 5.5. Fibres loaded with NaCl (by injection) show a prompt and sustained stimulation of the Na efflux when Na_e is replaced by Li⁺. A similar but less pronounced response is often seen with ouabain-poisoned fibres.
• 6.6. Injection of LiCl (e.g. a 2 M-solution), causes a 20% fall in Na efflux. Subsequent replacement of Na_e by Li⁺ fails to bring about a fall in the remaining efflux.
• 7.7. Itis concluded that the Na efflux in these fibres consists of a Na-Na exchange diffusion component which is not mediated by the Na-K pump and that its operation is interrupted by injecting Li⁺. The relative size of this component is about one-fifth and not one-half of the Na efflux.

     

Effect of warm acclimation on the cationic concentrations in the extracellular and intracellular body fluid of hibernating Rhagium inquisitor beetles

• 1.1. The effect of warm acclimation on the cationic concentrations of hibernating Rhagium inquisitor beetles was studied.
• 2.2. Following warm acclimation, the extracellular concentration of Mg²⁺ dropped from about 80 to about 50 mM. For Na⁺ and K⁺ the extracellular concentration remained constant at about 40 mM. The estimated intracellular concentration of Mg²⁺ was not affected by the warm acclimation, being about 80 mM in both groups of beetles. The corresponding concentration of Na⁺ increased from about 20 to about 30 mM, whereas for K⁺ there was no change, the concentration being about 140mM in both groups of beetles.
• 3.3. The marked drop in the extracellular concentration of Mg²⁺ might indicate that Mg²⁺ is involved in the regulation of the concentration of glycerol, which was reduced to zero during the warm acclimation.
• 4.4. The estimated equilibrium potentials of the respective ions might indicate that K⁺ is in electrochemical equilibrium across the cell membranes, whereas Na⁺ and to a lesser extent Mg²⁺ are actively transported out of the cells.
• 5.5. For Na⁺ there was a high positive equilibrium potential even in the cold acclimated beetles, indicating that Na⁺ has a high electrochemical potential difference across the cell membranes even at low temperatures. This is in agreement with the observation that hibernating insects are able to perform coorclinated walking immediately after heating to temperatures near zero.

     

Calcium uptake in the gorgonian Leptogorgia virgulata. The effects of atpase inhibitors

• 1.1. Longitudinally split or completely regenerated branch tips from Leplogorgia virgulata show no differences in calcium uptake between control and ouabain treatments. This indicates that there is no ouabain sensitive Na⁺, K⁺-ATPase involved in calcium uptake.
• 2.2. The tissue fractions of both regenerated and split branch tips show, at certain times, higher calcium uptake than control fractions. In the spicule fractions of these tips calcium uptake decreases in vandate treated specimens.
• 3.3. Pulse-chase experiments show an initial rapid release of calcium from the tips into surrounding seawater.
• 4.4. The results may suggest the presence of outwardly directed calcium pumps on the basal/lateral and apical plasma membranes of the epithelial cells. Outwardly directed calcium pumps may also be envisaged on the cell membranes of scleroblasts. In addition, pumps may move calcium into specific organelles of the scleroblasts en route to the spicule forming vacuoles.
• 5.5. These pumps are likely to be Ca²⁺-ATPase.

     

Isolation and partial amino acid sequence of A 78 kDa porcine gastrin-binding protein

• 1.1. A 78 kDa protein (p78) has been partially purified from washed membranes isolated from the corpus of porcine gastric mucosa. The purification was monitored by covalent cross-linking of iodinated [Nle¹⁵]-gastrin; 17.
• 2.2. A single N-terminal sequence extending for 33 amino acids was obtained from the p78 preparation. Partial sequences totalling 192 amino acids were also obtained from 14 tryptic and 3 Staphylococcal V8 peptides.
• 3.3. 10 peptides plus the N-terminal sequence were derived from a previously unsequenced protein which was distantly related to the product of the E. coli fadB gene (Baldwin G. S. (1993) Comp. Biochem. Physiol. 104B, 55–61). The remaining 7 peptides were derived from the gb-subunit of the gastric H⁺/K⁺-ATPase.
• 4.4. The gastrin-binding activity remained in association with p78, and could be separated from the P-subunit of the gastric H⁺K⁺-ATPase, during chromatography on tomato lectin-Sepharose.
• 5.5. We conclude that p78 binds gastrin, and is a novel member of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family of enzymes.

     

Sodium and potassium balance of captive meadow voles (Microtus pennsylvanicus) fed laboratory chow and vegetation diets

• 1.1. Mineral balance was studied in meadow voles (Microtus pennsylvanicus) maintained in the laboratory.
• 2.2. Urine and fecal Na⁺ contents of voles on low-Na⁺ diets were comparable to those reported for other herbivore species, but urine and fecal K levels were higher.
• 3.3. Voles approached Na⁺ balance (input = output) on diets with Na⁺ content as low as 56 ppm.
• 4.4. There was not a clearcut hypertrophy of the adrenal-gland zona glomerulosa in voles maintained on low-Na⁺ diets.
• 5.5. Plasma K content and bone water content were higher in voles maintained on high-Na ⁺ vegetation diets, suggesting expansion of extracellular fluid volume.

     

Sodium-dependence of amino acid transport by the nephridia of Sabella pavonina (annelida,polychaeta)

• 1.1. In the absence of sodium, the reabsorption rate of amino acid α-aminoisobutyric acid (AIB) by the nephridia of Sabella pavonina is reduced to 20% and the AIB accumulation in the cells is reduced to 10%. These results suggest the presence of sodium-dependant processes.
• 2.2. The observed processes are reversible when control conditions are re-established.
• 3.3. A minimum of 17mEq/l Na⁺ is required to restore the normal reabsorption rate.
• 4.4. The addition ofamiloride (10⁻³⁵ M) decreases the reabsorption rate, but to a lesser extent than the absence of sodium.

     

Crayfish retinular cells: Influence of extracellular sodium and calcium upon receptor potential

• 1.1. In crayfish, light stimulation of the retinular cells induces a depolarizing receptor potential.
• 2.2. Experiments were designed to determine the role of Na⁺ and Ca²⁺ on receptor potential during dark And light states.
• 3.3. Depolarization depends on Na⁺ and Ca²⁺ availability to the retinular cell.
• 4.4. Repolarization velocity and response duration depend on extracellular Ca²⁺ availability.
• 5.5. Light adaptation increases receptor potential dependence on calcium and sodium ions.
• 6.6. We analyse these results with respect to other invertebrate photoreceptors.

     

Cell surface labeling by iodine monochloride

• 1.1. Human red cell membranes were trace iodinated with [¹²⁵I]ICI and the distribution of label in membrane components examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 2.2. With intact erythrocytes over 84% of the bound iodine was associated with the membrane.
• 3.3. Two membrane components accounted for almost all of the label, band 3 and PAS-1.
• 4.4. Spectrin was not labeled in resealed ghosts.

     

Solubilization and characterization of functionally coupled Escherichia coli heat-stable toxin receptors and particulate guanylate cyclase associated with the cytoskeleton compartment of intestinal membranes

• 1.1. Particulate guanylate cyclase and receptors for E. coli heat-stable enterotoxin were solubilized from the rat intestinal cytoskeletal compartment using Lubrol-PX and KC1.
• 2.2. Thirty to forty percent of the ST receptor and guanylate cyclase activities were extracted from the lipid layer with Lubrol-PX alone.
• 3.2. Seventy percent of the remaining activities were solubilized from the cytoskeleton with Lubrol-PX and KCl.
• 4.3. Guanylate cyclase solubilized from either compartment exhibited similar reaction kinetics.
• 5.4. Both high- and low-affinity classes of ST receptors were solubilized from the lipid and cytoskeleton compartments.
• 6.5. In the presence of ATPγS, ST selectively activated the guanylate cyclase solubilized from the cytoskeleton compared to that solubilized from the lipid bilayer.
• 7.6. Crosslinking experiments demonstrated a preferential solubilization of the 130 kDa receptor subunit from the cytoskeleton and the 56 kDa subunit from the lipid bilayer.
• 8.7. Development of a procedure to solubilize ST receptors and guanylate cyclase from the intestinal membrane cytoskeleton will permit purification and further detailed studies of the coupling of these activities.