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.

     

Isolation of sarcolemmal membranes from cardiac muscle

• 1.1. Sarcolemmal membranes from cardiac tissue were isolated to a high degree of purity by brief extraction of homogenate with KC1 followed by 2 successive discontinuous sucrose density gradients of extracted particles.
• 2.2. Sarcolemmal membranes contained 40% adenylate cyclase, 22% guanylate cyclase and 20% ouabain-sensitive (Na⁺−K⁺) ATPase: contamination of sarcolemma by other intracellular membranes was negligible.
• 3.3. Electron microscopic examination of membranes showed presence of relatively empty vesicles of various shapes and sizes while electrophoretic analysis revealed about 20 protein bands of which 6 were prominent.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

Intestinal l-methionine transport in the cultured gilthead bream (Sparus aurata)

• 1.1. The influx and transepithelial movements of l-methionine and its effects on the electrophysiology and Na-Cl-transport in upper and lower intestine of the cultured fish, Spanis aurata, were measured.
• 2.2. The K_m and V_max of l-methionine influx into the tissues were higher in lower intestine than in upper intestine. A prominent diffusion-like transport component was also measured in both segments during influx experiments.
• 3.3. Net transepithelial fluxes of l-methionine (1 mM) were observed in both upper and lower intestine, this transport being Na⁺-dependent.
• 4.4. The two intestinal segments exhibited an electrical potential difference (PD) and a short circuit current (I_sc) serosa negative or near zero. Tissue conductance (G_t) was higher in posterior than in lower intestine.
• 5.5. Addition of l-methionine to the mucosal side of lower or upper intestine did not induce changes in PD in either part.
• 6.6. Isotopic fluxes of Cl⁻ or Na⁺ measurements under short circuit conditions showed that there were no net Cl⁻ or Na⁺ transport in either part.
• 7.7. l-Methionine additions to the mucosa did not induce changes in unidirectional fluxes of Cl⁻ or Na⁺ or in the (I_sc) in either the anterior or posterior intestine.

     

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.

     

Comparative studies of sodium transport and its relation to hydrostatic pressure in deep and shallow water gammarid crustaceans from Lake Baikal

• 1.1. Freshwater gammarids from 900–1400 m depths lose Na at 1 atm, 4°C, while related shallow water gammarids are near neutral Na balance.
• 2.2. Na⁺ influx rates are similar at 1 atm, 4°C, for abyssal and shallow water gammarids of similar weight.
• 3.3. Na⁺ efflux is faster for abyssal gammarids than for comparable shallow water gammarids.
• 4.4. Compressing abyssal gammarids to 90–140 atm increases Na⁺ influx rates enough to restore neutral Na balance, while in shallow water crustaceans, compression decreases Na⁺ influx.
• 5.5. Na⁺ influx rates in Baikalian gammarids vary with the 0.55 power of weight.
• 6.6. The equation F_{ma × t} = 1.3 × W^0.55 μEq/hr/animal applies to freshwater crustaceans over the weight range from 0.03 to 35 g.

     

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).

     

Human placental atp-diphosphohydrolase: Biochemical characterization,regulation and function

• 1.1. Kinetic and physico-chemical studies on human placental microsomal fraction confirmed that the ATPase and ADPase activities detected in this fraction correspond to the enzyme ATP-diphosphohydrolase or apyrase (EC 3.6.1.5). These include substrate specificity, and coincident M_r and pI values of both ATPase-ADPase activities.
• 2.2. This enzyme hydrolyses both the free unprotonated and cation-nucleotide complex, the catalytic efficiency for the latter being considerably higher.
• 3.3. Microsomal apyrase is insensitive to ouabain and Ap5A. The highly purified enzyme was only inhibited by o-vanadate, DBS and slightly by DCCD.
• 4.4. Apyrase seems to be a glycoprotein from its interaction with Concanavalin-A.
• 5.5. Preliminary studies on the essential amino acid residues suggest the participation of Arg, Lys and His residues, and discard the requirement of −SH, COO⁻, −OH, and probably also Tyr and Trp.
• 6.6. Two kinetic modulatory proteins of apyrase were detected in placental tissue. An activating protein was found in the soluble fraction and an inhibitory protein was loosely bound to the membranes.
• 7.7. The proposed in vivo function for apyrase is related to the inhibition of platelet aggregation due to its ADPase activity, which is supported by the direct effect on washed platelets and by its plasma membrane localization.

     

Effect of Li+ and Ba2+ on the electrocyte membrane-bound (Na+ + K+)-ATPase

• 1.1. Membrane-bound (Na⁺ + K⁺)-ATPase activity from the non-innervated and innervated faces of Electrophorus electricus (L.) electric organ, obtained by differential centrifugation, was measured using AChE as an enzyme marker for membranes derived from the post-synaptic area (fraction P₃) of the electrolyte.
• 2.2. The effect of Li⁺ and Ba²⁺ on (Na⁺ + K⁺)-ATPase activity of the two membrane fractions (P₂ and P₃) was analysed with respect to K⁺ and Mg²⁺ ions, after the I₅₀ estimation.
• 3.3. The kinetics of the reactions with these cations were investigated showing that Li⁺ inhibits P₂ uncompetitively and for P₃ presented a mixed type inhibition.
• 4.4. Ba²⁺ behaved as an hyperbolic mixed type inhibitor for P₂ and a linear mixed type inhibitor for P₃ fraction.

     

Possible involvement of adenylylation in the modification of a 26 kDa protein in rat parotid acinar cells

• 1.1. Adenylylation, a posttranslational modification of proteins, was investigated in saponin-permeabilized acinar cells of the rat parotid gland.
• 2.2. When cells were incubated with [2,8-³H]ATP, several proteins, including a 26 kDa protein in the particulate fraction, were labeled.
• 3.3. Upon incubation of cells with [α-³²P]ATP in the presence of cAMP and 3-isobutyl-lmethylxanthine, ³²P-labeling of the 26 kDa protein was observed.
• 4.4. After treatment with snake venom phosphodiesterase, [³²P]AMP was released from the 26kDa protein. Such release was not observed when cells were labeled with [γ-³²P]ATP.
• 5.5. The ³²P-labeling pattern of proteins with [α-³²P]ATP was clearly different from that with [adenylate-³²P]NAD⁺.
• 6.6. The results suggest that the 26 kDa protein is one of the adenylylation substrates in rat parotid acinar cells.

     

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.

     

Comparison of activities of several enzymes and protein constituents of oviducal and breast muscle of Japanese quail,Coturnix coturnix Japonica

• 1.1. Activities of several enzymes and protein constituents of magnum, isthmus, shell gland and breast muscle (pectoralis major) of Japanese quail, Coturnix coturnix japonica were compared.
• 2.2. The respective activity per g wet weight of lactate dehydrogenase, malate dehydrogenase and l-glycerol 3-phosphate dehydrogenase in pectoralis major was approx 20 times that of these enzymes in isthmus which showed the highest activity among oviducal tissues. On the other hand, the respective activity of lactate dehydrogenase was similar to that of malate dehydrogenase and was approx 10–20 times that of l-glycerol 3-phosphate dehydrogenase in each tissue.
• 3.3. Among NADPH-producing enzymes, NADP⁺-isocitrate dehydrogenase showed the highest activity in all tissues. The activity of malic enzyme was lowest in oviducal tissues, but was next to NADP⁺-isocitrate dehydrogenase in pectoralis major.
• 4.4. Sodium dodecyl sulfate polyacrylamide gel electrophoretic patterns of the whole homogenate, the supernatant and the precipitate fraction of magnum, isthmus, shell gland and pectoralis major showed tissue specific protein compositions. Proteins with molecular weight of 55,000, 70,000, 110,000 and 130,000 were observed in the respective precipitate (myofibrilar) fraction of magnum, isthmus and shell gland, but not in that of pectoralis major. It was noteworthy that the amount of myosin heavy chain of magnum was markedly less than that of other three tissues.

     

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.