Bioassay and proteolytic activity of digestive enzymes from octopus saliva

• 1.1. A bioassay for octopus saliva, based on detachment of crab dactylopodite flexor muscle under standard conditions, has been developed.
• 2.2. There is a direct relationship between increasing caseinolytic activity of saliva from Eledone cirrhosa and decreasing muscle detachment time.
• 3.3. Fractionation of saliva, using preparative isoelectric focusing, shows that muscle releasing activity is restricted to fractions containing proteins with high isoelectric points and maximum caseinase activity.
• 4.4. It is concluded that proteolytic enzyme(s) in octopus saliva selectively release crab muscle from attachment to the carapace.

     

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 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.

     

Na+ regulation and (Na++ K+)ATPase activity in the euryhaline fiddler crab Uca minax (Le conte)

• 1.1. Specific activity and kinetic characteristics of the (Na⁺ + K⁺)ATPase have been investigated in the gill epithelium of the hyper-hypoosmoregulator crab Uca minax.
• 2.2. (Na⁺ +K⁺)ATPase activity is shown to be at least three times higher in the posterior gills.
• 3.3. The kinetic study supports the hypothesis of the existence of two different (Na⁺ + K⁺)ATPases: the enzyme activity in the posterior gills could be involved in the transepithelial transport of Na⁺ while the activity of the anterior gills could be responsible for the intracellular regulation of Na⁺ and K⁺.
• 4.4. Significant and specific changes in (Na⁺ +K⁺)ATPase activity occur upon acclimation to media of various salinities.

     

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.

     

Characterization of NaK ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (de Man)

• 1.1. The specific activity of Na-K ATPase was determined from the microsomal preparation of gills dissected from adult Macrobrachium rosenbergii.
• 2.2. Maximal ATPase activity was achieved at a substrate concentration of 0.5 mM ATP.
• 3.3. Optimal enzyme activity was obtained at pH of 7.5.
• 4.4. The Arrhenius plot of Na-K ATPase activity revealed a marked discontinuity at 30°C. “Mg” ATPase activity did not exhibit a marked discontinuity.
• 5.5. The E_a for Na-K ATPase and “Mg” ATPase was 14.6 kCal/mole and 9.31 kCal/mole respectively. Q₁₀ values for Na-K ATPase was 2.34 and for “Mg” ATPase 1.65.
• 6.6. ATPase activity and gill homogenate protein concentration exhibited a linear relationship up to 130 μg protein/ml.
• 7.7. Na-K ATPase activity was inhibited by 10⁻³ M ouabain. It was equally inhibited by the removal of K⁺ from the reaction medium.

     

A comparison of the effect of alternating current electronarcosis,rectified current electronarcosis and chemical anaesthesia on the blood physiology of the freshwater bream Oreochromis mossambicus (peters)—III. The effect on the plasma electrolytes Ca2+, Na+ and K+ and on the osmotic pressure of the blood

• 1.1. The effects of alternating current electronarcosis, rectified current electronarcosis and chemical anaesthesia (benzocaine hydrochloride) on plasma electrolytes and on the osmotic pressure of the blood of the freshwater bream Oreochromis mossambicus were evaluated.
• 2.2. Plasma Ca²⁺, Na⁺ and K⁺ concentrations and the osmotic pressure of the blood were monitored over a period of 7 days.
• 3.3. The results showed that the different electrolytes respond differently to the different techniques.
• 4.4. Chemical anaesthesia exhibited the least effects on the parameters studied.

     

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.

     

Osmoregulation in the brook trout,Salvelinus fontinalis—I. Diel,photoperiod and growth related physiological changes in freshwater

• 1.1. Brook trout (Salvelinus fontinalis) raised from eggs under two photoperiod and two feeding regimes were tested for physiological changes preparatory for transition from freshwater to seawater. Size, age, growth rate, photoperiod, and diel rhythms were examined for possible influences on plasma osmolarity, [Na⁺], [Cl⁻], [K⁺], [Mg²⁺], thyroxine concentration, hematocrit, and gill Na⁺, K⁺-ATPase activity of brook trout in freshwater.
• 2.2. Significant diel cycles were found in plasma osmolarity, [Na⁺] and thyroxine concentration.
• 3.3. Significant size and/or age related changes occurred for plasma osmolarity, Na⁺], [K⁺] and hematocrit, but could explain little of their total variation (0.02 < r² < 0.18).
• 4.4. A sexually dimorphic response to photoperiod was observed in hematocrit for both mature and immature fish, with hematocrit of mature females declining in autumn and hematocrit of immature males increasing in autumn.
• 5.5. Gill Na⁺, K⁺-ATPase activity did not respond to photoperiod or feeding treatment and showed no change with size or age.
• 6.6. Plasma thyroxine levels responded to feeding and photoperiod treatment. There was a significant correlation between the percent mean difference in plasma thyroxine and the mean difference in growth rate between high and low feed fish (r² =0.51), suggesting a relationship between thyroxine and growth.

     

Hydrolysis of glycol chitin by chitinolytic enzymes

• 1.1. The hydrolysis of glycol chitin preparations by several β-N-acetylglucosaminidases was monitored colorimetrically with the potassium ferriferrocyanide reagent.
• 2.2. Glycol chitin samples from crab and insect sources varied considerably in chemical composition and susceptibility to enzymatic hydrolysis.
• 3.3. Insect endochitinase preferred crab glycol chitin as substrate while hen's egg white lysozyme preferred commercial glycol chitin.
• 4.4. Insect glycol chitin was well hydrolyzed by both enzymes.
• 5.5. Insect exochitinase did not digest glycol chitin.

     

Effect of repetitive cortisol and thyroxine injections on chloride cell number and Na+/K+-ATPase activity in gills of freshwater acclimated rainbow trout,Salmo gairdneri

• 1.1. Freshwater nonanadromous rainbow trout, Salmo gairdneri, were injected three times a week with either saline, 10μg cortisol/g, 1.0μg thyroxine/g or 10μg cortisol/g + 1.0μg thyroxine/g during a period of 28 days (12 injections). A separate group was derived as a subgroup from the thyroxine group on day 14 and received Cortisol + thyroxine from day 14 until day 28 (six injections).
• 2.2. Gill chloride cell number and Na⁺/K⁺-ATPase activity increased by cortisol treatment, the changes being significant on days 7 and 14, respectively.
• 3.3. Thyroxine treatment did not affect gill Na⁺/K⁺-ATPase activity or chloride cell number directly. Neither did it modify the stimulatory effect of cortisol on these parameters.
• 4.4. Muscle water decreased in cortisol-treated fish and increased in thyroxine-treated fish, while no changes were observed in the combined hormone groups.
• 5.5. No changes were observed in plasma chloride in any group during the experiment.
• 6.6. The results demonstrate a putative role of cortisol in stimulating hypo-osmoregulatory mechanisms and suggest that thyroxine is without a direct or a supportive effect for cortisol action.

     

Immunochemical and immunohistochemical determination of ubiquitin in molluscan (Mytilus edulis) smooth muscle

• 1.1. Immunochemical and immunohistochemical distribution of ubiquitin in the anterior byssus retractor muscle (ABRM) of Mytilus edulis was investigated.
• 2.2. In immunostaining, specific ubiquitin immunoreactivity was observed in the cross-sectioned ABRM, and was uniformly localized in this section.
• 3.3. The amount of free ubiquitin in the ABRM homogenate was 130 ± 4.6 ng/mg protein by western blot analysis, and ubiquitin conjugates were found at about 25, 29 and 200–230 kDa.
• 4.4. These findings were similar to those obtained in the skeletal muscle of rat.

     

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.

     

Osmoregulatory adjustments by three atherinids (Leptatherina presbyteroides; Craterocephalus mugiloides; Leptatherina wallacei) to a range of salinities

• 1.1. Patterns of osmoregulation were studied in three species of Swan river atherinids (Leptatherina presbyteroides, lower estuarine and marine; Craterocephalus mugiloides, mid estuarine; Leptatherina wallacei, upper estuarine) over a wide range of salinities.
• 2.2. The plasma Na⁺ concentration was elevated with an increase in salinity.
• 3.3. Haematocrit and body water content decreased with acclimation to higher salinity.
• 4.4. All three species of atherinids osmotically regulated over a salinity range greater than that which these fish are reported to occur in.

     

Modulation of active potassium transport by aldosterone

• 1.1. The role of aldosterone on active potassium transport across lizard colon under voltage-clamped conditions has been investigated.
• 2.2. Control colons exhibited no net potassium flux (J^k_net) despite of the existence of active opposite unidi ectional fluxes.
• 3.3. An important net secretory potassium flux was found in short-circuited aldosterone-stimulated colons.
• 4.4. Mucosal amiloride did not change (J^k_net) either in control or aldosterone-stimulated colons.
• 5.5. Luminal barium alters K ⁺ transport in a manner consistent with the presence of barium-sensitive conductances at the apical membrane of both control and aldosterone-treated colons.
• 6.6. The effects of ouabain and barium on control and aldosterone-induced potassium flows were consistent with a model involving basolateral uptake by an Na ⁺-K ⁺-ATPase and conductive exit across the apical membrane.
• 7.7. The stimulatory effect of aldosterone on potassium secretion is associated with parallel increases of both basolateral K ⁺ entry and the apical conductive pathway.

     

The effect of temperature on the acid-base status of the blood of the hatching quail (Coturnix c. japonica)

• 1.1. The ambient temperature of embryos of pipped eggs was reduced from 38 to 28°C for a period of 45 min.
• 2.2. The blood P_CO2 was lower and the blood more alkaline at 28°C than at 38°C.
• 3.3. At 28°C plasma [HCO₃⁻] ] was lower than predicted from the blood buffer line determined in vitro.
• 4.4. The plasma concentrations of strong ions and lactate were the same at both temperatures.
• 5.5. After the ambient temperature had been returned to 38°C for a period of 45 min, blood pH was more acidic than before cooling, but there was no difference in blood P_CO2.
• 6.6. The plasma [HCO₃⁻] was the same as that at 28°C and plasma [K⁺] was higher than before cooling.
• 7.7. The results arc discussed in relation to the factors affecting blood pH in embryos at this stage of development.

     

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.

     

Purification and some properties of elastase from hepatopancreas of king crab Paralithodes camtschatica

• 1.1. Elastase has been purified from the hepatopancreas of the king crab (Paralithodes camtschatica). Specific activity of the enzyme measured toward Suc-(Ala)₃-pNA and Boc-(Ala)₃-pNA was 926 and 3700 mUnits per mg of protein, respectively.
• 2.2. The enzyme is an anion protein (pI 4.5) with an approximate mol.wt of 28.5 kDa.
• 3.3. The enzyme exhibited a bell-shaped pH-dependence for the hydrolysis of Suc-(Ala)₃-pNA with a maximum at 8–8.5. Under these conditions the values of K_m and k_cat of the crab elastase are 4 mM and 4.75 s⁻¹, respectively.
• 4.4. The serine elastase is effectively inhibited by elastinal and diisopropylfluorophosphate.
• 5.5. It is shown that some salts except HgCl₂ activate the protease. In the presence of HgCl₂ with concentrations of 10 mM and higher, the crab elastase is inactive. SDS and Triton X-100 have no any effect on the activity of crab elastase.

     

Osmoregulation in the brook trout,Salvelinus fontinalis—II. Effects of size,age and photoperiod on seawater survival and ionic regulation

• 1.1. Brook trout (Salvelinus fontinalis) of a single genetic stock, and hatched at the same time, were raised under two photoperiod and two feeding regimes to obtain fish of the same age but with different sizes and photoperiod experiences. In 11 experiments over 1.5 firs, fish were gradually exposed to 32 ppt seawater for 20 days to investigate the ontogeny of salinity tolerance.
• 2.2. Daily changes in plasma osmolarity, [Na⁺], [Cl⁻], [K⁺], [Mg²⁺], thyroxine, hematocrit and gill Na⁺,K⁺-ATPase during adaptation to 10, 20 and 32 ppt were examined in one experiment.
• 3.3. Size was the primary determinant of seawater survival (r² = 0.77) the effect of size on seawater survival slowed after fish reached a fork length of 14 cm. The effect of age on seawater survival (r² = 0.65) was through its covariance with size.
• 4.4. Photoperiod affected seawater survival only through its influence on the timing of male maturation, which decreased salinity tolerance.
• 5.5. Regulation of plasma osmolarity, [Na⁺], [Cl⁻], [K²⁺], [Mg²⁺] and hematocrit in sea water increased linearly with size over the entire range of sizes (6–32 em).
• 6.6. Gill Na⁺,K⁺-ATPase activity after 20 days in seawater decreased with increasing size of brook trout, possibly reflecting decreased demand for active ion transport in larger fish.
• 7.7. Plasma thyroxine concentrations declined in seawater, but no definitive role of this hormone in seawater adaptation was found.
• 8.8. Size dependent survival and osmoregulatory ability of brook trout is compared to other salmonids and a conceptual model is developed.

     

Na+-K+ ATPase and carbonic anhydrase activities in larvae,postlarvae and adults of the shrimp Penaeus japonicus (Decapoda,Penaeidea)

• 1.1. Activities of Na⁺-K⁺ ATPase and carbonic anhydrase were measured through the early post-embryonic development of Penaeusjaponicus. In adults, only the Na⁺-K⁺ ATPase activity was measured.
• 2.2. ATPase activity was variable in the successive development stages. From zero in nauplii, the activity slightly increased in zoeae, and rose sharply in mysis stages 2 and 3.
• 3.3. A further significant increase in activity was noted at the transition from late mysis to early postlarvae, concomitant with a change from the larval osmoconforming pattern of osmoregulation to the postlarval and adult hyper-hyporegulating pattern.
• 4.4. The activity of Na⁺-K⁺ ATPase, measured in isolated cephalothorax, increased from PL3 to PL4 to its maximum value in PL5; at this stage, osmoregulatory capacity was fully efficient.
• 5.5. In young stages of P. japonicus, the variations in Na⁺-K⁺ ATPase activity appear correlated with the development of osmoregulatory ultrastructures, and with osmoregulation and salinity tolerance.
• 6.6. These results are discussed with regard to their ecological and physiological implications.
• 7.7. In adults, the activity of Na⁺-K⁺ ATPase was high in gills and epipodites and no activity was detected in branchiostegites. These results are related to the ultrastructure of these organs.
• 8.8. The activity of carbonic anhydrase did not change significantly in larval and postlarval stages.
• 9.9. From these results, it is proposed that the effector sites of osmoregulation are located in branchiostegites, pleurae and epipodites in postlarvae, and in epipodites and mainly in gills in adults.