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1.
The thermal inactivation of Ca2+ ATPase of scallop myofibrils (0.1 M KCl, pH 7.5) was found to be unaffected by the presence of Ca2+. Monomeric myosin content and salt solubility decreased much faster than Ca2+ ATPase inactivation in both Ca and EDTA media, which was well explained by faster denaturation of the rod portion than subfragment-1 of myosin. In contrast, when the myofibrils were heated at 0.5 M KCl, a slow decrease in salt solubility was observed, which was also explained by slow denaturation of the rod portion of myosin. Myofibrils from scallop smooth muscle showed the same denaturation pattern as those from adductor muscle. These results show that mollusk myosin is not always stabilized by Ca2+.  相似文献   

2.
Brook trout erythrocytes that were washed with and suspended in Ringer's solution with reduced glutathione (1.0 mM) maintained steady state cell volume for up to 24h, while those without the thiol-protective agent steadily shrank. Changes in cell volume (measured as packed cell volume, PCV) were evoked by acidic media (Ringer's at pH 6.8), hypoosmotic solutions (or both) and intracellular K+ and Cl concentrations were monitored over 4h. Acid-swollen cells failed to volume regulate or release K+ but had significantly elevated intracellular Cl Osmotically-swollen cells at pH 7.8 but not at pH 6.8 underwent regulatory volume decrease (RVD) and returned to initial levels in 2h, accompanied by release of K+ and Cl In contrast, osmotically-shrunken cells did not show regulatory volume increase. The regulatory volume decrease and concomitant K+ release were dependent on Cl implying a direct or indirect coupling of K+ to Cl transport in volume regulation. RVD was partially blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 0.1 mM), an anion exchange blocker, but was unaffected by amiloride (1.0 mM) which blocks Na+/H+ exchange. Amiloride and DIDS prevented the swelling response to low pH but had no effect on control cells, suggesting involvement of Na+/H+ and Cl/HCO3 exchanges in acid-induced cell swelling. Quinine (1.0 mM) a known blocker of K+ channels, exacerbated the osmotically-induced swelling but had little effect on the subsequent RVD and release of KCl. The results suggest that low extracellular pH inhibits neutral C-dependent K+ release and the resultant regulatory volume decrease in osmotically-swollen cells.  相似文献   

3.
Branchial plasma membranes from the freshwater cichlid teleostOreochromis mossambicus (tilapia) contain two Na+-dependent ATPases: Na+/K+ ATPase, and an amiloride-sensitive ATPase which is postulated to operate as a Na+/H+ (–NH4 +) ATPase. It is suggested that both enzyme activities are located in the basolateral membrane system of the chloride cells. K+ has opposing effects on the two enzymes: it stimulates Na+/K+ ATPase and inhibits Na+/H+ (–NH4 +) ATPase activity. Na+/H+ ATPase appears more sensitive to NH4 + at low concentrations than Na+/K+ ATPase and the stimulatory effect by NH4 + ions on the first enzyme could be important in facilitating NH4 + excretion by tilapia gills under physiological conditions.In vitro maximum stimulation by NH4 + is similar for the two enzymes (200%). In contrast to Na+/K+ ATPase, Na+/H+ ATPase activity is inhibited by supra-physiological (>20 mM) concentrations of NH4 +.  相似文献   

4.
The Australian freshwater fish, silver and golden perch, are increasingly being used for aquaculture. Addition of salt to water is commonly used in commercial aquaculture to reduce stress attributed to high ammonia concentrations. The activities in gill homogenates of ouabain-sensitive Na+/K+-ATPase and NEM-sensitive ATPases (as a measure of H+-ATPases) of silver and golden perch were measured after maintaining the fish in water containing different salt and ammonia concentrations. Six treatments were applied in a 2 × 3 factorial design: two salt treatments, low salt (LS) of 2.5 g l− 1 and high salt (HS) 5 g l− 1, and three ammonia treatments, no added ammonia (NA), low ammonia (LA), 3 mg total ammonia nitrogen (TAN) l− 1 and high ammonia (HA), 5 mg TAN l− 1. In both species, activity of Na+/K+-ATPase was lowest in fish kept in the LSNA treatment (7.4 ± 0.4 μmol Pi mg protein− 1 h− 1 for silver perch and 3.1 ± 0.6 for golden perch) and highest in the HSHA treatment (15.2 ± 1.0 μmol Pi mg− 1 protein h− 1 for silver and 8.4 ± 1.2 for golden perch). In both species there was a significant increase (P < 0.001) in Na+/K+-ATPase activity with increase in salt concentration and with an increase in ammonia concentrations. A significant interaction (P < 0.036) between salt and ammonia on Na+/K+-ATPase activity was observed in silver but not in golden perch. In contrast, the lowest activity for NEM-sensitive ATPase was observed in the HSNA treatment (1.0 ± 0.2 μmol Pi mg− 1 protein h− 1 for silver and 1.5 ± 0.4 for golden perch) and highest in LSHA treatment (2.9 ± 0.4 μmol Pi mg− 1 protein h− 1 for silver and 3.6 ± 1.2 for golden perch). In both species there was a significant decrease in NEM-sensitive ATPase activity with increase in salt concentration and an increase in activity with increase in ammonia (P < 0.003). In silver perch, a significant interaction between the treatments was observed (P < 0.02). The results suggest that in these species of freshwater fish the Na+/K+-ATPase has a role in salt and ammonia homeostasis and that the NEM-sensitive ATPases are more active in fish kept in water with a lower salt content. It is possible that the increase in ammonia resistance when salt is added to the environmental water in commercial aquaculture systems may be due to the effects of salt on gill Na+/K+-ATPase activity rather than the NEM-sensitive ATPases.  相似文献   

5.
Freshwater mussel Lamellidens marginalis showed a Ca2+-stimulated ATPase activity in the microsomal fraction of gill. Mg2+ was also found to be an activating cation for this ATPase enzyme. Ca2+ ATPase showed maximal activity at 40 °C and between pH 7.5–8.0. Substrate specificity of Ca2+ ATPase was highest with ATP, followed by GTP and other trinucleotides such as UTP, CTP and ADP also showed some amount of hydrolysis. Ca2+ ATPase showed slight inhibition with ruthenium red and sodium vanadate and is insensitive to sodium azide, ouabain, oligomycin B and phenylalanine. Heavy metals like Hg2+, Cu2+ and Zn2+ showed 50% inhibition of Ca2+ ATPase activity at concentrations of 0.25 mM, 0.5 mM and 1 mM, respectively whereas Cd2+ and Ni2+ showed 39% and 28% inhibition of enzyme activity at 1 mM concentrations.  相似文献   

6.
Mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1.39, L-malate: NAD+ oxidoreductase (decarboxylating)] was purified from herring skeletal muscle to a specific activity of 8.2 mol/min/mg. The purification procedure involved chromatography on DEAE-cellulose, Red Agarose and a Sephacryl S-300 with a final recovery of 38% of enzyme activity. This enzyme catalyzes the oxidative decarboxylation of malate in the presence of either NAD or NADP in the presence of Mn2+. Some kinetic characteristics of this enzyme were determined. The pH optimum of activity is 7.0. ATP was shown to be a competitive inhibitor with malate. The inhibition by ATP displayed hyperbolic competitive kinetics with a Ki (ATP) of 0.28 mM in the presence of NAD and 0.75 mM in the presence of NADP. Fumarate reversed ATP inhibition.In vivo, regulation of NAD(P)-dependent malic enzyme might respond to changing levels of mitochondrial ATP and fumarate with the enzyme undergoing kinetic activation by an increase in the concentration of mitochondrial fumarate which could reverse enzyme inhibition by ATP.  相似文献   

7.
Soft water acclimated (Ca2+ 0.02 mM; Na+ 0.03 mM; K+ 0.01 mM; pH 7.0), cannulated brown trout (Salmo trutta) were exposed to various pH and aluminium (Al) regimes (pH 7.0, pH 5.0, pH 5.0 plus Al: 50, 25, and 12.5 g l–1) for up to 5 days in order to determine (i) the sublethal concentration of Al at pH 5.0 for this species (ii) their ionoregulatory and respiratory status. No mortality or physiological disturbances were evident at pH 7.0 or pH 5.0. All trout died within 48 h at pH 5.0 in the presence of Al at 50 g l–1 and 67% died over the 5 day period at pH 5.0 in the presence of Al at 25 g l–1. Fish at these lethal Al concentrations showed significant decreases in arterial blood oxygen content (CaO2) but no changes in plasma osmolarity or the concentrations of plasma Na+, K+ and Cl. Physiological disturbance was more marked at the 50 g l–1 Al concentration. The surviving fish at 25 g l–1 showed few signs of physiological recovery while continually exposed to this regime. No fish died during the exposure to water of pH 5.0 containing 12.5 g l–1 Al, but physiological disturbance was still apparent. These sublethally-stressed trout showed a transient decline in the plasma concentrations of Na+ and Cl–1. Although CaO2 decreased, recovery was evident. The data suggest that in the brown trout, environmental Al concentration is as important as pH and calcium concentration in determining the physiological status of the fish.  相似文献   

8.
The composition of seminal plasma and metabolism of sperm of the cyprinid fishAlburnus alburnus were investigated. Statistically significant correlations were found between motility parameters and seminal fluid osmolality, pH, Na+, K+ and protein levels (negative correlations: % immotile spermatozoa-Na+, K+; positive correlations: % motile spermatozoa-osmolality, pH, Na+, K+, protein; % linear motile spermatozoa-pH protein; swimming velocity of spermatozoa-pH, Na+, protein). Spermatozoan motility and ATP metabolism and glycolysis were correlated as indicated by measurement of ATPase, pyruvate kinase, adenylate kinase and lactate dehydrogenase activity. The physiological meanings of these correlations and their possible significance for quality control of semen are discussed.Abbreviations used ACP acid phosphatase - ADP adenosine diphosphate - AK adenylate kinase - ALP alkaline phosphatase - ASPAT aspartate aminotransferase - ATP adenosine triphosphate - ATPase magnesium dependent adenosine triphosphatase - CRPO creatine phosphate - -GLU \-D-glucuronidase - ICDH isocitrate dehydrogenase - LDH lactate dehydrogenase - PK pyruvate kinase  相似文献   

9.
The spermatozoa of oviparous fish, such as feral carp (Cyprinus carpio), are immotile in the presence of semen plasma or isotonic solutions, and to obtain good motility, they must be diluted with suitable medium. The objective of this study was to identify the best activating solution for feral carp sperm. Sperm motilities were compared in the new activating solution (a): (50 mM NaCl, 30 mM KCl, 30 mM Tris, pH = 8.5) and activating solution (b): (50 mM NaCl, 40 mM KCl, 30 mM Tris, pH = 8.5) based on effect of pH with everyone of Na+ and K+ ions versus four other activating solutions Billard’s saline solution, Poupard’s saline solution, distilled water and hatchery water that is routinely used for extending carp semen. Our results showed that maximum total motility period and percentage of motile sperm were seen in selected saline solution (a). The present study describes an activating solution that prolongs feral carp sperm motility.  相似文献   

10.
Branchial activities of Na+,K+-ATPase (ouabain sensitive), Mg2+ ATPase (ouabain insensitive) and kinetic analysis of high and low affinity Ca2+ ATPase were measured inAnguilla anguilla that had been acclimated to demineralized water (DW, Ca < 10 M), freshwater (FW, Ca = 2 mM), and Low calcium freshwater (L-Ca, Ca = 0.9 mM). Na+,K+-ATPase activity decreased while ouabain insensitive activity increased when ambient Ca2+ decreased. Two kinetic forms of Ca2+ ATPase could be resolved in each environmental condition. The stimulation coefficients of both sites or enzymes were not affected by ambient Ca2+ concentrations. The maximal velocity of both the high and the low affinity Ca2+ ATPase was increased when external Ca2+ was decreased during acclimation. The low affinity Ca2+ ATPase and the Mg2+ stimulated enzyme could be a non specific enzyme accepting either Ca2+ or Mg2+. Results are compared with previous results in the literature and in relation to the branchial morphology and ionic exchanges in fish.  相似文献   

11.
To evaluate the quality of frozen surimi, the ATPase assay system was applied to pH stat. Surimi homogenate was prepared without washing procedures as a material for the measurement. Among the possible compounds frozen surimi contains, Mg ion slightly inhibited Ca-ATPase activity, while sugars or polyphosphate did not. Thus, the surimi homogenate was directly used for ATPase assay. The quality of surimi samples with various commercial grades were analyzed by measuring Ca-ATPase activity and protein content to obtain ATPase total activity. SA grade surimi showed high total activity, and was clearly distinguished from the second grade ones by the total Ca-ATPase activity. The specific Ca-ATPase activity contributed to the total activity more than the protein content. These results demonstrated that employment of surimi homogenate and the pH stat method made the total activity measurement of surimi easy and rapid.  相似文献   

12.
The activities of mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase, were examined in sea bass (Dicentrarchus labrax L) liver. The activities of the three enzymes were studiedin vitro in relation to the influence of protein content, time of incubation, pH, temperature, mevalonate, ATP and Mg++ concentration. Protein content in the assay medium affected the three enzymes differently. Mevalonate kinase, mevalonate 5-phosphate kinase, and mevalonate 5-pyrophosphate decarboxylase activities were linear up to 0.2, 0.4 and 0.8 mg protein, respectively. With respect to the time course studies, the enzymes also behaved differently. Mevalonate kinase activity increased over forty minutes, reaching a plateau thereafter, while mevalonate 5-phosphate kinase and decarboxylase increased over the entire assay period. All the three enzymes showed a maximum in activity at pH 7.5. The effect of reaction temperature showed that phosphorylation increased to maximum around 35°C for mevalonate kinase and 30°C for mevalonate 5-phosphate kinase while decarboxylation rates remained constant well until 30°C temperature decreasing afterwards. The enzymes behaved differently as a function of mevalonate concentration. Mevalonate 5-phosphate formed was maximal when the initial mevalonate concentration was 272 M, whereas mevalonate 5-pyrophosphate and CO2 were formed maximally at mevalonate concentrations of 136 M and 68M, respectively. Optimal ATP concentration in the medium was 3 mM for decarboxylase and 6 mM for kinases, and Mg++ requirements varied from 4 mM for decarboxylase to 6 mM for kinases.  相似文献   

13.
The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during short-term storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n?=?5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg?1. Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0–9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.  相似文献   

14.
The present study investigated motility, acrosome reaction, fertility and cryobiological parameters of the semen of the sterlet, Acipenser ruthenus L. Sperm motility persisted for about 4 min in water, and the main swimming type was the linear motion. Motility was prolonged at osmolalities of 12.5 mosmol kg?1 and in the presence of magnesium ions, while calcium had no effect. Also a pH in the range of 7.0–9.0 had no effect on ` motility. At osmolalities of 25–50 mosmol kg?1 the sperm motility was partly inhibited, at osmolalities of 100 mosmol kg?1, completely and irreversibly. In 50 mosmol kg?1 solutions with 2.5–5 mM L?1 KCl the motility inhibition was total, but reversible. The acrosome reaction was not induced by one of the described solutions, but the percentage of spermatozoa with reacted acrosomes was low (<20%) and highly variable in all experiments. The optimal extender base for cryopreservation was a solution consisting of 50 mM L?1 NaCl, 5 mM L?1 KCl, 10 mM L?1 Tris (pH 8.5). From the tested cryoprotectants only dimethylsulphoxide (DMSO) and methanol provided sufficient cryoprotection. After freezing and thawing, the motility rates and swimming velocities were higher with DMSO than with methanol. However, the fertility was very significantly reduced with DMSO (10.3±0.5%) while with methanol fertilization rates in a similar range (32.7±4.4%) as with fresh semen (33.90±0.8%) could be obtained. Optimal freezing conditions for sterlet semen were in the vapour of liquid nitrogen 3–5 cm (?95°C to ?85°C) above its surface, the optimal thawing conditions at 25°C for 30 s. The acrosome reaction was not induced by these cryopreservation protocols.  相似文献   

15.
The effects of NaCl supplementation (0.0%, 2.5%, 5.0%, 7.5% and 10.0% dry weight of a basal diet) on growth, gill histological alterations and osmoregulation of juvenile cobia reared in low‐salinity water (5 g L?1) were assessed. At the end of the experiment, gills were sampled for Na+, K+‐ATPase activity determination and histological evaluation. In all treatments, no mortality was observed. Results showed that dietary NaCl supplementation did not alter growth. At the highest supplementations (7.5% and 10.0%), juvenile cobia showed higher feed intake and feed conversion ratio. Na+, K+‐ATPase activity was higher in fish fed the diet without salt supplementation than in those fed with NaCl‐supplemented diets. The number of chloride cells significantly increased with increasing dietary salt level, being 2.5‐fold higher in fish fed with 10.0% NaCl supplementation (41 cells mm?2) than in those from the non‐supplemented fed group (16 cells mm?2). These findings indicate that dietary salt supplementation stimulated chloride cell proliferation paralleled with a reduction in the gill Na+, K+‐ATPase activity, suggesting a possible decrease in energy consumption associated with osmoregulation. However, the suggested energy sparing did not have a significant impact on juvenile cobia growth.  相似文献   

16.
In vivo and in vitro protein digestibility of 0% to 60% soybean meal protein (SBM) substitution from fishmeal protein was conducted for bagrid catfish Mystus nemurus juveniles. Seven experimental diets containing 35% protein and 15% lipid were fed to bagrid catfish to determine the in vivo protein digestibility. In vitro methods were determined using pH stat, pH shift, spectrophotometric assay and sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) with different enzyme mixtures (crude intestinal extract of bagrid catfish, Lazo 1‐enzyme, Hsu 3‐enzyme, Saterlee 4‐enzyme). In vivo and in vitro protein digestibility decreased with increased SBM protein substitution with highest tolerance level of 10% (in vivo). pH stat and pH drop methods showed the highest degree of hydrolysis (DH) and relative protein digestibility (RPD) using Saterlee 4‐enzyme system. However, pH stat method showed highest correlations (r2 = 0.9263) with the in vivo results using crude intestinal enzyme extract compared to other enzyme systems. The highest correlation of the in vitro methods using crude intestinal enzyme extracts of bagrid catfish was determined using spectrophotometric assay (r2 = 0.9284) followed by pH stat (r2 = 0.9263), SDS‐PAGE (r2 = 0.8348) and pH drop (r2 = 0.6777). All the in vitro methods tested are suitable to rapidly determine protein digestibility for bagrid catfish except for pH drop.  相似文献   

17.
Several physical and chemical parameters of angiotensin-converting enzyme (ACE) were determined using a spectrophotometric assay of gill tissue homogenates from rainbow trout. This assay is based on the evolution of free hippuric acid via enzymatic cleavage of histidyl-leucine from the synthetic substrate hippuryl-l-histidyl-l-leucine (HHL). Piscine ACE exhibited enzymatic and kinetic properties similar to those reported for the partially purified mammalian enzyme. Proteolytic activity was both temperature and pH dependent and demonstrated hyperbolic kinetics with an apparent Km of 2.5 mM. Hydrolysis of HHL was activated by Cl at concentrations between 20 mM and 100 mM. Captopril (1 × 10–6 M) and MK-422 (1 × 10–6 M) blocked trout gill ACE activity, however, EDTA was inhibitory only at high concentrations (1 × 10–3 M). These results demonstrate that trout ACE is functionally similar to mammalian ACE and that the spectro-photometric assay for ACE developed by Cushman and Cheung can be applied to analysis of converting enzyme activity in fish tissue homogenates.  相似文献   

18.
Gill Na+-K+ ATPase and carbonic anhydrase activities were measured, on a fortnightly basis, from February to July, in 0+ age Atlantic salmon (Salmo solar), hatched and reared in a freshwater experimental station in Covas, northern Portugal. Plasma osmolarity and ionic composition were also measured. Gill Na+-K+ ATPase activity increased slowly until April (15–19 moles Pi mg prot–1 h–1). From April to late May there was a great increase in activity (19–32 moles Pi mg prot–1 h–1) followed by a sharp decline in June (15 moles Pi mg prot–1 h–1). In contrast, carbonic anhydrase activity decreased significantly from early April to early June (170-70 moles p-nitrophenol mg prot–1 h–1) and increased in late June, suggesting the existence of a compensatory mechanism for the changes in Na+-K+ ATPase activity. Plasma osmolarity and sodium concentration showed lower levels during the period of high ATPase activity. On the other hand, plasma calcium concentrations showed an increase during the same period (3.47–5.98 mm1–1 of plasma). A transitory decrease in osmolarity and plasma sodium and chlorine concentrations occurred in March, prior to the surge in Na+-K+ ATPase activity, suggesting that the physiological changes, characteristic of parr-smolt transformation can be a consequence of this loss of freshwater osmoregulatory capacity.  相似文献   

19.
The nitrate threshold concentration in rearing water of African catfish (Clarias gariepinus) was assessed. Female African catfish with an initial mean (SD) weight of 154.3 (7.5) g were exposed to 0.4 (Control), 1.5, 4.2, 9.7 and 27.0 mM nitrate for 42 days. Mean (SD) plasma concentrations of nitrate increased from 71 (29) to 6623 (921) μM at the highest ambient nitrate level. Mean (SD) plasma nitrite concentration ranged from 1.2 (0.5) to 7.9 (9.0) μM. Haematocrit, plasma concentrations of non‐esterified fatty acids (NEFA), cortisol, glucose, lactate, osmolality, gill morphology and branchial Na+/K+‐ATPase activity were not affected. Feed intake and specific growth rate were significantly reduced at the highest nitrate concentration. We advise not to exceed a water nitrate concentration of 10 mM (140 mg L?1 NO3‐N) to prevent the risk of reduced growth and feed intake in African catfish aquaculture.  相似文献   

20.
ABSTRACT

Due to negative health effects of high sodium intake, it is recommended to reduce the daily salt intake by around 50%. To reduce the sodium content, sodium salts can be exchanged with potassium or magnesium salts. The effect of sodium, potassium, and magnesium chlorides on extractability of proteins from fresh and frozen haddock muscle and minces was studied. Salting with KCl and MgCl2 instead of NaCl changed protein extractability. The highest solubility of the proteins was achieved using Na+. However, at low concentrations, extractability in K+ and Mg2+ is on the same level as Na+, showing that partial substitution of NaCl with KCl or MgCl2 is possible. Freezing affected the structure of tissue and protein properties, resulting in decreased amount of salt soluble proteins.  相似文献   

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