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.

     

Localization of the glucocorticoid receptor in rat liver cells: Evidence for plasma membrane bound receptor

• 1.1. The cytoplasmic glucocorticoid receptor of rat liver cells is in part recovered in the plasma membrane fraction.
• 2.2. After in vivo administration of [³H]dexamethasone, 0.35% of the radioactivity recovered is bound on plasma membranes.
• 3.3. Dexamethasone also binds in vitro specifically to plasma membranes. Expressed as fmol/mg protein, binding of dexamethasone to plasma membranes is comparable to binding to the soluble cytoplasmic fraction (cytosol).
• 4.4. Using polyclonal antibody to the glucocorticoid receptor and the indirect immunofluorescence technic, an intense decoration of the plasma membranes is observed, denoting a high concentration of glucocorticoid receptor on plasma membranes.
• 5.5. The localization of the receptor on plasma membranes could be of potential importance for its interaction with agents (mitogens, growth factors) initially acting on the cell membrane, regulating subsequent cell proliferation and growth at the level of the cell nucleus.

     

Isolation of chromaffin cell thapsigargin-sensitive Ca2+ store in light microsomes from bovine adrenal medulla

• 1.1. A subcellular fractionation procedure for bovine adrenal glands was designed with the aim to study the biochemical properties of Ca²⁺ stores in chromaffin cells.
• 2.2. The thapsigargin-sensitive compartment of Ca²⁺ stores was found to be highly enriched in a light microsomal fraction (LMF) on a 15–30% linear sucrose gradient, and was found to be essentially devoid of contamination by plasma, mitochondrial or secretory granule membranes.
• 3.3. A Ca²⁺-pumping ATPase was identified in this LMF as a 97 kDa protein forming an acid-stable, Ca²⁺-dependent, thapsigargin-sensitive phosphorylated intermediate upon incubation with [γ-³²P]ATP, suggesting this protein to represent a SERCA-3 isoform of Ca²⁺ ATPases.
• 4.4. A major 162 kDa protein, previously demonstrated in the isolated chromaffin cells, was enriched in the LMF, distributing on sucrose gradients in parallel with the thapsigargin-sensitive Ca²⁺ uptake.
• 5.5. LMF appears to represent a part of the thapsigargin-sensitive Ca²⁺ store of chromaffin cells, and should be useful for further studies of the store properties at the subcellular and molecular level.

     

The isolation of a plasma membrane-rich fraction from the skeletal muscle of two species of marine crab,Carcinus maenas and Cancer pagurus

• 1.1. A rapid method for the isolation of a plasma membrane-rich fraction from crab leg muscle, with high purity and yield recovery was developed.
• 2.2. The method is based on sodium iodide extraction of the crude homogenate, followed by centrifugation on Percoll self-creating gradient.
• 3.3. (Na⁺K⁺)ATPase and alkaline phosphodiesterase I were used as marker enzymes for the plasma membrane and revealed levels of purification of approximately 13-fold and yields recovery of the total activity in the crude muscle homogenate of approximately 18%, for both species of crab studied.

     

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.

     

In vivo responses in plasma osmolality,and intracellular water and solute concentrations in flounder (Platichthys flesus) heart ventricles under acclimation to fresh and sea water

• 1.1. Flounders transferred abruptly from sea to fresh water displayed a gradual decrease in plasma osmolality for 5–6 days (10–15 mOsm daily). When returned to sea water the osmolality increased to the original level within 1 day.
• 2.2. Heart ventricle cell water content remained unchanged during the acclimations, except for a temporary 1.4% reduction within the first 4 hr of sea water acclimation.
• 3.3. During acclimation to sea water intracellular K⁺ increased rapidly in parallel with plasma osmolality. During fresh water acclimation, however, cellular K⁺ decreased rapidly in the first day only, whereas plasma osmolality decreased further.
• 4.4. Cellular taurine remained unchanged during the initial 4 days of fresh water acclimation and then declined 32% within the next 3 days. Upon retransfer to sea water, cellular taurine increased gradually to its original level in the course of 7 days.

     

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.

     

Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.

     

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.

     

The effect of mercury and aluminum on sodium-potassium-Mg2+ dependent-adenosine triphosphatase activity of Electrophorus electricus (L.) electrocyte

• 1.1. Na⁺,K⁺-ATPase, which mediates the active transport of Na⁺ and K⁺ across the plasma membrane, is found in equivalent amounts in both plasma membranes of the electrocyte, the anterior, non-innervated (fraction P₂) and the posterior, innervated (fraction P₃) obtained by differential centrifu gation of Electrophorus electricus (L.) electric organ.
• 2.2. The kinetic effects of Hg²⁺ and A1³⁺, described as neurotoxic metals, on the Na⁺,K⁺-ATPase activity of the two membrane fractions (P₂ and P₃) were analysed with respect to Na⁺ and K⁺ ions, after the I₅₀ estimation of each metal.
• 3.3. Mercury is a potent Na⁺,K⁺-ATPase inhibitor in the nanomolar range. In all cases, it behaved as a mixed partial hyperbolic inhibitor.
• 4.4. Aluminum was shown to be a poor enzyme inhibitor. Changing the K⁺ concentration, it behaved as a mixed linear inhibitor (P₂ fraction) and as a non-essential mixed activator (fraction P₃). Aluminum behaved as a partial hyperbolic inhibitor for both P₂ and P₃ fractions with respect to Na⁺ concentration.
• 5.5. The observation of the variable kinetic behaviour of P₂ and P₃ led us to attribute these differences to the Na⁺,K⁺-ATPase electrocyte isoenzymes which occur in different proportions in these fractions (Gomes-Quintana et al., 1992 Comp. biochem. Physiol.103B/3 623–628).

     

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.

     

In vitro Ca deposition by rat matrix vesicles: Is the membrane association of alkaline phosphatase essential for matrix vesicle-mediated calcium deposition?

• 1.1. Phosphatidylinositol phospholipase C (PI-PLC) treatment of rachitic rat matrix vesicles (MVs) released about 80% of membrane-bound alkaline phosphatase (ALP), AMPase, PPiase into the media.
• 2.2. About 20% hydrolytic activity was not released from MV membranes by PI-PLC treatment.
• 3.3. SDS-polyacrylamide gel electrophoresis and Western blot analysis showed only one immunoreactive protein corresponding to the molecular weight of ALP present in the soluble fraction after PI-PLC treatment.
• 4.4. The specific activity of the released ALP was at least 5-fold higher than the residual activity.
• 5.5. After PI-PLC treatment, MVs also demonstrated an 80% reduction of AMP- or βGP-dependent calcium deposition.
• 6.6. The soluble fraction containing 80% of ALP activity was unable to support calcium deposition. The mixing of the soluble and insoluble fractions after PI-PLC treatment failed to fully restore calcium-depositing activity.

     

Electrolyte changes,cell volume regulation and hormonal influences during acclimation of rainbow trout (Salmo gairdnerii) to salt water

• 1.1. Rainbow trout were acclimated to salt water (1.5, 2.0 or 3.0%, which means 40, 60 or 85% concentrated sea-water) and the electrolyte, glucose and cortisol concentrations of the plasma as well as the extra- and intracellular muscle space, the muscle electrolyte concentrations and the ATPase activity were analysed.
• 2.2. Plasma osmolality, Na⁺, Ca²⁺ and Mg²⁺ concentrations of the plasma had a maximum at 24 hr after the start of acclimation when acclimated to 3.0% salt water. Plasma osmolality, Na⁺ and Mg²⁺ concentrations were significantly higher during the whole acclimation time when exposed to 3.0% salt water.
• 3.3. Variations and regulations of ECS and ICS were clearly demonstrated. The intracellular electrolyte concentrations were also maximal at 24 hr.
• 4.4. The plasma glucose level was just slightly elevated, but the cortisol level clearly indicated a stress response at 24 hr.
• 5.5. The activity of gill Na-K-ATPase increased during the acclimation time.
• 6.6. The regulatory processes in trout during acclimation to salt water are compared with those occurring in tilapia and carp.

     

The effect of eyestalk ablation on growth,haemolymph composition and gill Na+, K+-ATPase activity of Penaeus monodon juveniles

• 1.1. Eyestalk unablated and unilaterally ablated Penaeus monodon juveniles had survival rates after 5 months of 75–72.5 and 67.5–60%, respectively.
• 2.2. Unilaterally ablated shrimps had significantly higher (P < 0.05) growth rate than unablated shrimps.
• 3.3. Eyestalk-ablatement resulted in a decrease in the haemolymph sodium concentration and an increase in the potassium and calcium concentration of shrimps.
• 4.4. The osmolarity of haemolymph and total protein concentration of unablated shrimps were demonstrated to be higher than those of unilaterally ablated shrimps.
• 5.5. The eyestalk-ablated shrimps possess higher total ATPase and Na⁺,K⁺-ATPase activities in the gill than those of unablated shrimps.

     

Ionic dependence and vanadate sensitivity of (Na+, K+)-ATPase isolated from branchial sac of ascidians

• 1.1. Ion dependence and vanadium-induced inhibition on branchial sac ATPase in five species of ascidian Phlebobranchiata (vanadium-accumulating) and Stolidobranchiata (iron-accumulating) were studied.
• 2.2. The ATPase was obtained from the microsomal fraction, which was prepared from each ascidian branchial sac.
• 3.3. The ATPase was dependent on Mg²⁺ and activated by exogenous Na⁺ + K⁺.
• 4.4. Ouabain inhibited the ATPase activity in vitro, 10 μM to 100 μM vanadate, in vitro, suppressed the (Na⁺, K⁺)-ATPase.

     

Sea-water tolerance in freshwater-resident arctic charr (Salvelinus alpinus)

• 1.1. Freshwater-resident Arctic charr acclimated for 2 months at 8°C, 15% were divided into four experimental groups in July and exposed to 1 and 8°C in 15 and 34% salinity.
• 2.2. Only slight changes in gill Na-K-ATPase activity, blood plasma osmolality and blood plasma concentrations of Cl⁻ and Mg²⁺ were found for the fish exposed to 1 or 8°C in brackish water.
• 3.3. When exposed to sea-water at 8°C, an increase in osmolality and in concentrations of Cl⁻ and Mg²⁺ took place during the first 2–3 days, after which it levelled off.
• 4.4. If exposed to sea-water at 1°C, however, marked increases were found for all parameters measured and all the fish were dead within 5 days of exposure.
• 5.5. These results show that freshwater-resident Arctic charr—if acclimated to brackish water—can survive in sea-water during summer if the environmental temperature is not too low.

     

A study on the rna polymerase activities in bovine thyroid nuclei isolated in isotonic sucrose,hypertonic sucrose or in anhydrous glycerol media: Influence of the isolation procedure on the electrophoretic pattern of acid soluble nuclear proteins

• 1.1. After differential pelleting of bovine thyroid bound RNA polymerase II was the more enriched enzyme activity in the nuclear fraction, and coincided best with the DNA profile.
• 2.2. The RNA polymerase I + III activity was compared in nuclear fractions isolated either in 0.25 M sucrose (wet tissue) or in anhydrous glycerol (lyophilized tissue) or in 2.4 M sucrose (lyophilized tissue).
• 3.3. Although the nuclei were more resistant to the isolation porcedure in glycerol, more proteins were extracted by that procedure than during the isolation in 2.4 M sucrose.
• 4.4. With the 2.4 M sucrose method a twofold enrichment of RNA polymerase I + III activity in respect to DNA occurred in the nuclei pointing to an exclusive localization of these activities within the nucleus.
• 5.5. Using the same isolation procedure the different classes of histones were better resolved upon polyacrylamide gelelectrophoresis.

     

The possibility that Ca2+-ATPase from the plasma membrane-rich fraction of bovine parotid gland is ecto-Ca2+-dependent nucleoside triphosphatase

• 1.1. Parotid plasma membrane nonpump low-affinity Ca²⁺-ATPase, which possesses high-affinity (Ca²⁺ + Mg²⁺ )-ATPase activity, was characterized.
• 2.2. Purified Ca²⁺-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67–93% of ATP) and nucleoside diphosphates, ADP. GDP, IDP, CDP, TDP (12–40% of ATP) but not AMP and p-NPP.
• 3.3. The maximum activities of Ca²⁺- and (Ca²⁺ +Mg²⁺ )-ATPases were obtained in the presence of 1 mM and 0.13 μ M Ca²⁺, respectively.
• 4.4. The K_m values for Ca²⁺ in Ca²⁺- and (Ca²⁺+ Mg²⁺ )-ATPases were 0.2 mM and 22 nM. respectively.
• 5.5. The activities of both Ca²⁺- and (Ca²⁺ + Mg²⁺ )-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction.
• 6.6. These features suggest that Ca²⁺-ATPase is an ecto-Ca²⁺-dependent nucleoside triphosphatase.

     

Tetanus responses under rapid bath solution change: Electrotonic depolarization of transverse tubules may release Ca2+ from sarcoplasmic reticulum of Rana japonica skeletal muscle

• 1.1. Single skeletal muscle fibers were transferred from a normal Ringer solution to Na⁺ ion free solution, and vice versa, and tetanus responses were recorded immediately after the transfer.
• 2.2. Fractional tetanus tension recorded immediately after the displacement from the Na⁺ ion free solution to normal Ringer solution was dependent on fiber diameter.
• 3.3. Diffusion of Na⁺ ions along the transverse tubules was simulated [apparent diffusion constant was 3.11 × 10⁻⁶ (cm²/s)].
• 4.4. Our results suggest that the electrotonic spreading of membrane potential, caused by an action potential in the transverse tubules, could release Ca²⁺ ions from sarcoplasmic reticulum.

     

Plasma membrane: Can its structure and function be modulated by dietary fat?

• 1.1. Compositional analysis of plasma membranes from rats fed nutritionally adequate diets different in fatty acid composition establishes that fundamentally different dietary fat intake results in alteration in structural lipid composition of plasma membranes in brain, liver and the intestinal mucosa.
• 2.2. Dietary differences in fatty acid intake altered the fatty acyl tail composition of plasma membrane phospholipids in brain, liver and intestinal mucosa.
• 3.3. Diet altered the phospholipid profile observed in brain synaptosomal and liver plasma membrane.
• 4.4. Feeding high vs low polyunsaturated to saturated fat diets for 7 days altered the fatty acid composition of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and mono-glucosylceramide isolated from plasma membrane of the intestinal mucosa