The partial double Donnan—A simple alternative interpretation of the transmembrane ionic distribution

• 1.1. The pump-leak hypothesis of transmembrane ion distribution implicitly poses the operational immobilization of a fraction of intracellular potassium and extracellular sodium.
• 2.2. Subtraction of operationally immobilized ion fractions from total intracellular potassium and extracellular sodium concentrations leads to Donnan ratios of the mobile fractions consistent with the transmembrane distribution of the chloride ions and potentials derived from the constant field equation.
• 3.3. The predictions of this simple approach agree with the experimental observations regarding ionosmotic processes in several types of aquatic animal cells.

     

Short-term osmotic responses of cells and tissues of the sea anemone,Condylactis gigantea

• 1.1. Cells of tentacles and body wall of the sea anemone Condylactis gigantea behaved as simple osmometers during 5hr exposure to 50, 67, 83, 100 and 125% sea-water.
• 2.2. All intracellular water appeared to be osmotically active.
• 3.3. Cell sodium, chloride and total osmolyte content remained invariable, with taurine decreasing and potassium increasing as sea-water concentration was reduced.
• 4.4. Tissues, as a whole, exhibited a pseudoregulatory response to changes in salinity as the large and osmotically inert extracellular space buffered volume changes to a considerable extent.

     

Kinetic studies of the transphosphorylation reactions catalyzed by alkaline phosphatase from E. coli: Hydrolysis of p-nitrophenyl phosphate and o-carboxyphenyl phosphate in presence of tris

• 1.1. Transphosphorylation of p-nitrophenyl phosphate and o-carboxyphenyl phosphate to Tris, has been studied at alkaline and acid pH.
• 2.2. The rate of release for all reactions products was Tris-dependent for both substrates, with a slight maximum for phenol at alkaline pH. These dependences have been analyzed from a mechanistic standpoint.
• 3.3. Individual constants of rate of a simple transphosphorylation mechanism have been determined.
• 4.4. At high Tris concentrations (> 1.0 M) a slight competitive inhibition has been observed.
• 5.5. Inhibition in NH₄⁺-NH₃Cl buffer has been found at alkaline pH but not at acid pH. It would therefore seem that the non-protonated NH₂ group of Tris is responsible for inhibition.
• 6.6. The results suggest the formation of complexes between Tris and the enzyme. Other possible alternatives are also analyzed.

     

Are coelenterate cells permeable to large anions?

• 1.1. Substitution of chloride by isethionate, a bulky anion, did not modify the intracellular cation or water content in cells of tentacles, and only slightly decreased sodium content in body wall cells of the coelenterate Condylactis gigantea, in contrast with the appreciable reductions expected in the case of impenneant anions.
• 2.2. As isethionic acid is a strong acid, the salt should be almost totally ionized at the pH of seawater (8.6) and at the presumably close to neutral intracellular pH. Therefore, the anion, rather than the undissociated acid, would appear to be the permeating species.
• 3.3. Isethionate ion appears to distribute across the cell membrane as does chloride: according to the transmembrane potential difference.

     

Driving forces for the uphill transport of amino acids into epidermal brush border membrane vesicles of the sea anemone,Anemonia sulcata (Cnidaria,anthozoa)

• 1.1. The transport of amino acids into membrane vesicles prepared from epidermal tentacle tissue of the sea anemone, Anemonia sulcata, depends on an electrochemical potential difference caused, e.g. by sodium chloride gradients.
• 2.2. Potassium or choline chloride gradients energized the transport less effectively than sodium chloride gradients. Both Na⁺-ions and Cl⁻-ions were required for the amino acid transport.
• 3.3. The uphill transport of amino acids along the downhill movement of driver ions (sodium chloride gradient conditions) was characterized by an overshoot; under sodium chloride equilibrium conditions, however, an accumulation of amino acids within the vesicles could not be measured.
• 4.4. Potassium diffusion potentials in combination with valinomycin indicated that hyperpolarization (vesicle inside negative) and hypopolarization (vesicle inside positive) enhanced or depressed the accumulation of amino acids within the vesicles.
• 5.5. Being at the phylogenetic base of the Eumetazoa, cnidarians show characteristics for the transmembrane transport of amino acids comparable to those established for vertebrates.

     

Trypanosoma brucei: A quick method for separating blood-stream trypomastigotes from infected blood by differential osmotic lysis

• 1.1. The degree of rat erythrocyte lysis and immobilization of Trypanosoma brucei in infected blood by buffered hypotonie solutions of sodium chloride and sucrose was studied.
• 2.2. At 0.3% sodium chloride solution 98% hemolysis of erythrocytes was achieved while 95% of the original bloodstream trypomastigotes survived and were found to be motile and viable for biochemical study.
• 3.3. Further increase in the concentration of sodium chloride above 0.3% revealed an increase in the immobilization of trypanosomes and a decrease in the erythocyte hemolysis.
• 4.4. Bloodstream trypomastigotes have been prepared by differential osmotic lysis of infected blood in 0.3% sodium chloride solution and used for studying their metabolism.

     

Examination of water balance and ionic regulation in the earthworm,Eisenia foetida

• 1.1. Weight change after submerging the earthworm into water varied remarkably according to the environmental humidity in which animals were placed before submergence.
• 2.2. Pretreatment with physiological saline solution before submergence in water gave stable values for the ionic concentrations of the body fluid.
• 3.3. Brain removal caused decrease of both sodium and chloride ion concentrations and increase of potassium ion concentration of the coelomic fluid when animals were submerged in water.
• 4.4. Although brain replacement failed, action of a brain hormone is suggested to regulate the decrease of both sodium and choride ions and increase of potassium ion of the coelomic fluid to normal level when animals were submerged in water.

     

The effect of reduced salinity on tissue and plasma composition of the dogfish,Squalus acanthias

• 1.1. Dogfish (Squalus acanthias) were acclimated to reduced salinities and their plasma, muscle tissue and erythrocytes subsequently analysed.
• 2.2. Decrease in the osmolarity of the plasma was principally due to a fall in urea concentration and a significant fall in the concentrations of sodium and chloride.
• 3.3. Changes in the muscle and erythrocytes in dilute media were a decrease in urea, potassium, sodium and chloride concentrations.
• 4.4. The concentrations of the free amino acids in the muscle and the red blood cells decreased more than would be expected by the movements of water only.
• 5.5. The results were discussed in relation to the regulation of cellular volume and the involvement of the free amino acid pool of the tissues in this process.

     

The effect of osmotic and ion-osmotic stresses on the blood and urine composition and urine flow of rainbow trout (Salmo gairdneri)

• 1.1. Changes in urine and plasma concentrations (sodium, potassium, magnesium, calcium and total osmotic) and urine production were determined in fish exposed to various concentrations of an ionically active substance, sodium chloride, and a non-electrolyte, mannitol, as well as freshwater.
• 2.2. Responses occurred for the most part over a short crisis period preceeding establishment of new stable conditions.
• 3.3. It was shown that plasma homeostasis was not maintained in response to changing ion-osmotic and osmotic gradients.
• 4.4. Urinary osmotic and ionic concentrations were unaffected and urine production was shown to be inversely related to the external concentration.
• 5.5. It is suggested that ionic shifts between body compartments are an important aspect of ion-osmotic adaptation.

     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

     

The chemical composition of the follicular fluid of the teleost Clinus dorsalis (Perciformes: Clinidae)

• 1.1. The osmolarity and pH of the follicular fluid was determined and analyses of total glucose, total lipids, total proteins, amino acids, urea, sodium and potassium carried out.
• 2.2. The mean osmolarity of the follicular fluid was found to be 325 mOsm/kg and the mean pH was 7.9.
• 3.3. The embryotrophe was rich in lipids (1092.39 mg/100 ml) and amino acids with the amino acid concentration exceeding normal values for human plasma.

     

Oxygen binding of intact coelomic cells and extracted hemoglobin of the echiuran Urechis caupo

• 1.1. The oxygen affinity of Urechis caupo coelomic cells is the same in normoxic and in hypoxic animals. There is no Bohr effect between pH 6.8 and 8.0.
• 2.2. The oxygen affinity of intact coelomic cells is the same as that of extracted, stripped hemoglobin. The oxygen binding properties of stripped hemoglobin are not affected by 1 mM ATP, IMP, or hydrogen ions between pH 6.8 and 8.0, nor do they clearly show cooperativity. The heat of oxygenation. ΔH, = −13.1 kcal/mol between 10 and 25 C.
• 3.3. Although U. caupo coelomic cell hemoglobin is tetrameric and intracellular, it apparently exhibits neither heterotropic nor homotropic interactions.

     

Water diffusion,ion transport and lipid composition of the skin of Rana cyanophlyctis Schneider under osmotic stress

• 1.1. Analysis of the total lipid content (TL), components of the neutral lipid fraction (NLF), phospholipid fraction (PLF), recordings of electrical potential differences and diffusional permeability were carried out in the skin of the aquatic frog Rana cyanophlyctis subjected to in vivo salt stress (0.9 sodium chloride) for different durations (0, 1, 3 and 7 days).
• 2.2. A general decrease of skin TL and of components of the NEE and PEE was observed.
• 3.3. Stoichiometric ratios for skin PLF components under initial salt stress of different durations reveal an increase of the ratios of sphingomyelin and phosphatidyl choline after osmotic stress.
• 4.4. The diffusional permeability of water increased following exposure to salt stress of I, 3 and 7 days duration.
• 5.5. The transepithelial potential difference measured in vitro after a salt stress of 3 days was considerably higher than the controls.

     

The high intracellular ph buffering of hagfish (Eptatretus cirrhatus) dental plate retractor muscle cannot be attributed to the known histidine-related compounds present in other vertebrates

• 1.1. Intracellular pH buffering capacity of hagfish (Eplatretus cirrhatus) dental plate retractor muscles is among the highest reported for any vertebrate muscle.
• 2.2. Over 80% of the pH buffering capacity of hagfish retractor and myotome muscle is due to components other than proteins and phosphate.
• 3.3. The muscles have less than 0.5 μmol/g wet weight of l-histidine, and lack l-l-methyl histidine, l-3-methyl histidine and the histidine-containing dipeptides anserine, carnosine and ophidine.
• 4.4. Instead, they contain an unidentified low molecular weight acid-soluble compound to which the high pH buffering capacity can be attributed.

     

Anuran amphibia which are not acclimable to high salt,tolerate high plasma urea

• 1.1. The capacity of five anuran Amphibians (Bufo viridis B. regularis, Rana ridibunda, Hyla arborea and Pelobates syriacus) to acclimate to NaCl and urea solutions was investigated.
• 2.2. All species could be acclimated to relatively high concentrations of urea solutions, while only Bufo viridis and Hyla arborea could be acclimated to 500 mOsm/kg or higher NaCl solutions.
• 3.3. The plasma urea concentration in B. viridis and H. arborea was elevated to levels over 140 mmol/1.
• 4.4. The sum of plasma sodium and chloride concentrations did not increase over 400 mmol/l in any species.
• 5.5. Urine osmolality, which was normally low, increased, but never exceeded the plasma osmolality.
• 6.6. In the urea acclimation conditions, urine electrolytes diminished, similarly in all species in this study.
• 7.7. It is concluded that anuran Amphibians can tolerate high plasma urea concentrations, but only those species which can elevate it, either through retention or net synthesis, can be acclimated to high salt solutions.

     

Biochemical studies in a eunicid (polychaete) of the littoral of São Paulo,Brazil

• 1.1. The sodium, potassium, chloride, calcium, magnesium, phosphorus, sulphur, copper, iron, total carbon dioxide, uric acid, creatinine, urea, glucose, erythrocruoin, nitrogen, total iodine, protein-bound iodine, total lipids, triglycerides, alkaline phosphatase activity, acid phosphatase activity and copper oxidase activity contents of the blood of the giant Polychaete, Eunice sp., were determined.
• 2.2. The osmolarity of the blood was 997 mOsm/1 and the pH was 6·49, a very low value. The bicarbonate concentration estimated by the Henderson-Hasselbach equation was 4·70 mM/1.
• 3.3. The values of the sea water, sediments, water contained in the tube, tube, cuticle, muscle and faeces are also given.
• 4.4. The chemical composition of the mucus was determined.

     

A simple and reliable method for the purification of Pseudomonas aeruginosa phospholipase C produced in a high phosphate medium containing choline

• 1.1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-P_i basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite.
• 2.2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate.
• 3.3. The peak containing the PLC activity revealed a single protein after SDS-PAGE.
• 4.4. The method could also be applied to purify PLC produced in a low-P_i complex medium. The resultant preparation was not homogeneous.
• 5.5. The molecular weight for both PLC preparations was about 70 kDa.
• 6.6. Both PLC used phosphatydilcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent K_M of 25 mM for p-nitrophenylphosphorylcholine.
• 7.7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100.
• 8.8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HP_l-BSM plus choline but not the enzyme from the LP_l-CM.

     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

The effects of extracellular-and intracellular-pH on transmitter mobilization and fractional release in Helix pomatia

• 1.1. Transmitter mobilization and fractional release were studied in Helix pomatia. The right palliai nerve was stimulated and a synaptic potential was recorded in cell F76 in the right parietal ganglion.
• 2.2. The extra- and intra-cellular pH were changed with Tris-maleate, CO₂ or (NH₄)₂SO₄.
• 3.3. The time constant for the monoexponential part of mobilization decreased with reduced intracellular pH. Only a fraction of this effect could be related to an increase in the intracellular Ca-activity.
• 4.4. Fractional release was reduced in low external pH, but was increased in low intracellular pH. Fractional release is affected more by changes in internal pH than external pH.

     

Changes in composition and proteolytic enzyme activities of artemia during early development

• 1.1. The proximate composition, total and free amino acids, and proteases of Artemia nauplii were determined during early development.
• 2.2. Moisture increased from 71.0% to 80.8%, crude protein decreased from 13.2% to 8.8%, crude fat and ash varied slightly.
• 3.3. The total amino acids decreased. Free amino acids changed in three patterns.
• 4.4. Trypsin, chymotrypsin, carboxypeptidase A, B and cathepsin B and C increased in activity. The activity of trypsin was lower, while cathepsin B and C were the highest.
• 5.5. The protease activities were maximal at pH 7.5 and 8.0, and at 45°C on casein.
• 6.6. The optimal pH for carboxypeptidase A was 4.0, for carboxypeptidase B was 4.5, for trypsin and chymotrypsin were 7.0–7.5. The protease(s) active at pH 9.0–9.5 were to be determined.