Effects of temperature,pH, and heavy metals (copper,zinc) upon proteinase activities in digestive tract mucosa of typical and facultative piscivorous fish

Species-specific differences and effects of natural (temperature, pH) and anthropogenic (zinc, copper) factors upon activities of casein-and hemoglobinlytic proteinases have been studied in the gut mucosa in pike (Esox lucius), zander (Stizostedion lucioperca), burbot (Lota lota) and perch (Perca fluviatilis). It is revealed that the intestine mucosa proteinase activities differ considerably from those ones in the fish stomach mucosa and that studied factors affect these activities to different extents. The stomach mucosa proteinases are most tolerant to the effects of studied factors, which is especially true for pike. The least tolerant are the intestine caseinlytic proteinases (except for the enzymes in perch). The effects of temperature, pH, and heavy metals depend on fish species, factors’ combinations (mono-factor, bi-factor, or multi-factor action) and on factor variability.     

虎纹蛙消化道淀粉酶和脂肪酶活力研究

以酶学分析方法研究了虎纹蛙消化道淀粉酶和脂肪酶的分布以及pH和温度对这两种消化酶活力的影响。结果表明：在各自生理pH值条件下,虎纹蛙消化道不同部位淀粉酶活力大小顺序依次为前肠〉中肠〉后肠〉食道〉胃,胃和肠淀粉酶最适pH值分别为8．6和7．0,最适温度分别为35℃和40℃。脂肪酶活力大小顺序依次为中肠〉后肠〉前肠〉胃〉食道,各部位之间差异显著（P〈0．05）,胃和肠脂肪酶的最适pH值均为9．0,最适温度分别为50℃和55℃。     

The Effect of pH on the Supply of Digestive Hydrolases in Fish with Different Feeding Habits

Journal of Ichthyology - The effect of pH on the activity of peptidases and glycosidases functioning in the intestine and the supply of these enzymes in five fish species from the Rybinsk Reservoir...     

Effect of copper on liver key enzymes of anaerobic glucose metabolism from freshwater tropical fish Prochilodus lineatus

We investigated the effect of copper on liver key enzymes of the anaerobic glucose metabolism (hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK; lactate dehydrogenase, LDH) as well as of the pentose pathway (glycose-6-phosphate dehydrogenase, G6PDH) from the fish Prochilodus lineatus. The fish were acclimated at either 20 degrees C or 30 degrees C at pH 7.0, transferred to water at pH 4.5 or 8.0, and exposed to 96 h-CL(50) copper concentrations. Copper accumulation in liver was higher in fish acclimated at 20 degrees C and maintained in water pH 8.0. Three-way analysis of variance revealed a significant effect of temperature on all enzymes, a significant effect of pH on all enzymes except for PK, and a significant effect of copper on only PFK, and LDH in pH 4.5 at 20 degrees C and, at 30 degrees C, on PFK and PK at pH 4.5 and 8.0, HK at pH 4.5 and G6PDH at pH 8.0. There were significant interactions between treatments for many enzymes. These changes suggest that the activity of enzymes in question is modified by a change in ambient water. At least at 30 degrees C, the overall reduction in the glycolytic enzyme activities of copper-exposed fish seems to reduce energy availability via glucose metabolism, thereby contributing to enhance copper toxic effects.     

Effect of pH on the proteinase activities in the intestine mucosa,chyme, and enteral microbiota in the piscivorous fish differing in their ecological traits

The effect of pH on the activity of proteinases of intestine mucosa, chyme, and enteral microbiota was studied in three piscivorous fish species of the Rybinsk Reservoir differing in their ecological traits: pike Esox lucius, burbot Lota lota, and zander Sander lucioperca. In all fish species, studied pH of ten is optimal for the functioning of proteinases of intestine mucosa; in chyme proteinases, optimal pH values vary from six to ten. Optimal pH for functioning of proteinases of enteral microbiota is seven for zander and pike; pH of nine, for burbot. High activity of chyme and microbiota proteinases within the diapason of pH values of six to nine is a characteristic of burbot. Relative activity of proteinases of intestine mucosa in all fish species is not more than 15% of the maximal activity; that of chyme and enteral microbiota is lower than 40% in zander, close to 50% in burbot, and 80 and 50%, respectively, in pike. It is suggested that diversity of the patterns of pH-dependence of enteral microbiota proteinases relates to the specificity of microbiota in various ecological zones of the reservoir (littoral, sublittoral, and bathyal).     

Partial characterization of digestive proteases in Totoaba (Totoaba macdonaldi)

ABSTRACT

The objective of this study was to characterize the digestive proteases of totoaba (Totoaba macdonaldi). Fish were sacrificed to obtain the multienzymatic extracts from the stomach and intestine, and determine the stability and optimum pH and temperature values. Residual activity and number of isoforms were determined with some inhibitors. Optimal pH of stomach proteases was 2, with stability above 100% at that same pH. Optimum pH of intestinal proteases was between 9 and 11, with stability above 100% between 8-12. Optimum temperature for stomach proteases was 35°C and remained highly stable, while optimum temperature for intestinal proteases was 45°C, with high stability between 35-55°C. Pepstatin A totally inhibited acid protease activity and revealed a single band. SDS-PAGE zymogram revealed 8 bands in the intestine, where phenanthroline inhibited 80% of the total activity. The digestive capacity of T. macdonaldi is characteristic of a strict carnivore, similar to other marine fish species.     

Physiological pH values in the digestive tract of perch <Emphasis Type="Italic">Perca fluviatilis</Emphasis> from different habitats

Physiological pH values are determined in the gastrointestinal tract of the perch Perca fluviatilis from lakes Chany and Baikal. Values of pH are detected to vary significantly along the digestive tract. Such factors as a segment of the digestive tract, presence/absence of food, season of the year, and habitat affect pH values. Environmental temperature is the main factor affecting pH values that is confirmed by the decrease in pH values upon water temperature rise in spring, lower pH values in summer compared to other seasons, and lower pH in fish from the waterbody (Lake Chany) with higher temperature of water.     

Final stages of carbohydrate metabolism in a cestode Eubothrium rugosum (Cestoda: Pseudophyllidea)

Characteristics of final stages of carbohydrate metabolism in a cestode Eubothrium rugosum from the burbot intestine have been studied. It is found out that in the course of in vitro incubation the worms produce lactic acid to the environment with a concomitant acidification of the medium. The rate of pH changes and lactate production eventually fell during the incubation. The medium pH being the result of helminth's metabolism varies within the normal physiological limits of the host intestine pH and pH optima for its basic digestive enzymes.     

Ammonia in estuaries and effects on fish

This review aims to explore the biological responses of fish in estuaries to increased levels of environmental ammonia. Results from laboratory and field studies on responses of fish to varying salinity and their responses increased ammonia will be evaluated, although studies which examine responses to ammonia, in relation to varying salinity, pH and temperature together are rare. In a survey of British estuaries the continuous measurement of total ammonia showed values that ranged from background levels increasing up to c. 10 mg N l^?1 although higher values have been noted sporadically. In outer estuaries pH values tended to stabilize towards sea water values (e.g. c. pH 8). Upper reaches of estuaries are influenced by the quality of their fresh waters sources which can show a wide range of pH and water quality values depending on geological, climatic and pollution conditions. In general the ammonia toxicity (96 h LC₅₀) to marine species (e.g. 0·09–3·35 mg l^?1 NH₃) appears to be roughly similar to freshwater species (e.g. 0·068–2·0 mg l^?1 NH₃). Ammonia toxicity is related to differences between species and pH rather than to the comparatively minor influences of salinity and temperature. In the marine environment the toxicity of ionized ammonia should be considered. The water quality standard for freshwater salmonids of 21 μg l^?1 NH₃–N was considered to be protective for most marine fish and estuarine fish although the influence of cyclical changes in pH, salinity and temperature were not considered. During ammonia exposures, whether chronic or episodic, estuarine fish may be most at risk as larvae or juveniles, at elevated temperatures, if salinity is near the seawater value and if the pH value of the water is decreased. They are also likely to be at risk from ammonia intoxication in waters of low salinity, high pH and high ammonia levels. These conditions are likely to promote ammonia transfer from the environment into the fish, both as ionized and unionized ammonia, as well as promoting ammonia retention by the fish. Fish are more likely to be prone to ammonia toxicity if they are not feeding, are stressed and if they are active and swimming. Episodic or cycling exposures should also be considered in relation to the rate at which the animal is able to accumulate and excrete ammonia and the physiological processes involved in the transfer of ammonia. In the complex environment of an estuary, evaluation of ammonia as a pollutant will involve field and laboratory experiments to determine the responses of fish to ammonia as salinity and temperature vary over a period of time. It will also be necessary to evaluate the responses of a variety of species including estuarine residents and migrants.     

Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii

The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.     

The isozymes of glucose-phosphate isomerase (GPI-A2 and GPI-B2) from the teleost fish Fundulus heteroclitus (L.)

The fish, Fundulus heteroclitus (L.), like most advanced teleosts, possesses duplicate loci for the glycolytic enzyme, glucose-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). The locus for the GPI-A2 (where GPI represents glucose-phosphate isomerase) isozyme is preferentially expressed in anaerobic tissues such as white skeletal muscle, while GPI-B2 predominates in aerobic tissues like liver and red muscle. We questioned whether this tissue specificity would be reflected in unique structural and functional characteristics of the respective isozymes. Consequently, an analysis of the two isozymes was undertaken. The enzymes were purified by a combination of ion-exchange chromatography and isoelectric focusing. Each isozyme was characterized as to native and subunit molecular weight, isoelectric pH, and susceptibility to thermal denaturation. Both were dimeric enzymes, with native molecular masses of 110 kDa. The isoelectric pH values for GPI-A2 and GPI-B2 were 7.9 and 6.4, respectively. Differences were apparent in thermal stability, i.e. GPI-A2 was more stable than GPI-B2. Kinetic properties were investigated as a function of both pH and temperature. The Km values for fructose 6-phosphate (Fru-6-P) differed between the isozymes at low pH, but no significant differences were observed at higher pH. The inhibition constant (Ki) for 6-phosphogluconate (6-P-gluconate) was pH dependent. GPI-A2 was slightly more sensitive to 6-P-gluconate inhibition than GPI-B2 between pH 7.0 and 8.5. The Km for Fru-6-P was temperature dependent for the GPI-B2 isozyme, but relatively temperature independent for GPI-A2 between 10 and 35 degrees C. The Ki for 6-P-gluconate was temperature dependent for both isozymes. The Ki values for GPI-A2 were consistently lower than those for GPI-B2. Energies of activation differed between the two isozymes by 4.4 kcal with GPI-A2 having the lower value. While delta G values were identical for the isozymes, their delta H and delta S values differed significantly. The structural and kinetic differences that exist between the glucose-phosphate isomerase isozymes appear to be tailored to the unique metabolic demands of the tissues in which these Gpi loci are expressed.     

Protein digestion and proteolytic activity in the digestive tract of an omnivorous cyprinid

1. In adult and juvenile roach (Rutilus rutilus) feeding on meal worms or grass and acclimated to temperatures between 6 and 20 degrees C the following variables were determined: pH, protein and proteolytic activity of gut fluid and faeces, food consumption, duration of gut passage and efficiency of protein assimilation. 2. Proteolytic enzymes of fish are very stable against autolysis but they disappear in posterior portions of the intestine, suggesting the existence of a pinocytotic process. 3. In herbivorous roach as well as in adult carnivorous roach feeding at 20 degrees C this results in very low proteolytic activities in the faeces, whereas in juvenile fish and in adults feeding on meal worms at lower temperatures, the process of reabsorption seems to be less efficient. 4. Daily production of proteases as well as "daily proteolytic duration" are higher in herbivorous than in carnivorous roach. 5. For the same amount of protein consumed, fish feeding on grass require 10 times higher proteolytic activities than fish feeding on meal worms.     

Disaccharidase activities in camel small intestine: biochemical investigations of maltase-glucoamylase activity

Disaccharidases (maltase, cellobiase, lactase, and sucrase), alpha-amylase, and glucoamylase in the camel small intestine were investigated to integrate the enzymatic digestion profile in camel. High activities were detected for maltase and glucoamylase, followed by moderate levels of sucrase and alpha-amylase. Very low activity levels were detected for lactase and cellobiase. Camel intestinal maltase-glucoamylase (MG) was purified by DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of camel small intestinal MG4 and MG6 were estimated to be 140,000 and 180,000 using Sephacryl S-200. These values were confirmed by SDS-PAGE, where the two enzymes migrated as single subunits. This study encompassed characterization of MGs from camel intestine. The Km values of MG4 and MG6 were estimated to be 13.3 mM and 20 mM maltose, respectively. Substrate specificity for MG4 and MG6 indicated that the two enzymes are maltase-glucoamylases because they catalysed the hydrolysis of maltose and starch with alpha-1,4 and alpha-1,6 glycosidic bonds, but not sucrose with alpha-1,2 glycosidic bond which was hydrolyzed by sucrase-isomaltase. Camel intestinal MG4 and MG6 had the same optimum pH at 7.0 and temperature optimum at 50 degrees C and 40 degrees C, respectively. The two enzymes were stable up to 50 degrees C and 40 degrees C, followed by strong decrease in activity at 60 degrees C and 50 degrees C, respectively. The effect of divalent cations on the activity of camel intestinal MG4 and MG6 was studied. All the examined divalent cations Ca(2+), Mn(2+), Mg(2+), Co(2+) and Fe(3+) had slight effects on the two enzymes except Hg(2+) which had a strong inhibitory effect. The effect of different inhibitors on MG4 and MG6 indicated that the two enzymes had a cysteine residue.     

Growth depression in socially subordinate rainbow trout Oncorhynchus mykiss: more than a fasting effect

To assess the effects of subordinate social status on digestive function, metabolism, and enzyme activity in salmonid fish, juvenile rainbow trout Oncorhynchus mykiss were paired with size-matched conspecifics (<1.5% difference in fork length) for 5 d. Fish that were fasted for 5 d and fish sampled directly from the holding tank were used as control groups. Both subordinate and fasted fish experienced significant decreases in intestine mass (P = 0.043), and the gall bladder showed marked and significant changes in both size (P = 0.004) and appearance. These findings suggest that the negative effect of social subordination on digestive function reflects in large part a lack of feeding. Hepatic phosphoenolpyruvate carboxykinase activity was significantly higher in subordinate fish relative to dominants, whereas subordinate hepatic pyruvate kinase activity was significantly lower; activities of both enzymes were significantly correlated with plasma cortisol concentrations and behavior scores. Dominant-subordinate differences in the activities of these enzymes were eliminated by administration of the glucocorticoid receptor antagonist RU486, underlining a role for circulating cortisol in eliciting the differences. Significant increases relative to control fish were also detected in red and white muscles from subordinate fish in the activities of protein catabolic enzymes (aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase). These differences occurred in the absence of any change in plasma free amino acid or ammonia concentrations, supporting an enhanced turnover of amino acids in muscle in subordinate fish. The results support the hypothesis that changes in metabolism, beyond those elicited by low food consumption, may be responsible at least in part for the low growth rates typical of subordinate fish and that these changes may be related specifically to circulating cortisol levels in subordinate fish.     

Utilisation of male Arctic capelin and Atlantic cod intestines for fish sauce production--evaluation of fermentation conditions

The present paper shows that the fish by-products male Arctic capelin and Atlantic cod intestines can be utilized as raw materials for the production of high value fish sauce for human consumption. By supplementing minced capelin with 5-10% enzyme-rich cod pyloric caeca, a good recovery of fish sauce protein (60%) was obtained after 6 months of storage. Although, the proteases present in cod pylorus caeca are cold adapted enzymes, a storage temperature of 26 degrees C gave a higher fish sauce recovery than storage at 21 degrees C. Initial alkalification of samples to pH 8 accelerated the tissue solubilisation and gave a better fish sauce recovery without adveresely affecting the pH of the final product. Addition of small amounts of calcium had no significant effects on either protease activity or protein recovery in the fish sauce fraction.     

General review of fish protein hydrolysates

Fish protein hydrolysates are generally considered to be the liquefied products obtained from fish by the action of proteolytic enzymes under accelerated conditions of digestion. The proteolytic enzymes which are used for the digestion of fish proteins must be active either above the survival temperature of spoilage bacteria or outside the range of pH which would support their growth. By suitable selection of proteolytic enzymes and conditions for hydrolysis some control over the digestion can be achieved to give a range of products. This paper reviews the work done here and elsewhere on fish protein hydrolysates and discusses their potential for making more effective use of the fish resources.     

Postprandial changes in pH and enzyme activity from the stomach and intestines of Tilapia rendalli (Boulenger, 1897), Oreochromis mossambicus (Peters, 1852) and Clarias gariepinus (Burchell, 1822)

The ability to utilise dietary components differs among fish species. Digestive enzymes may be used to determine the efficiency of the digestive process. In this study, the activities of the digestive enzymes in Tilapia rendalli, Oreochromis mossambicus and Clarias gariepinus were explored. Protease, amylase, lipase and cellulase activities were measured in different parts of the digestive tract of the three fish species. The pH dynamics along the digestive tract were monitored. In all fish species, the presence of food led to a reduction in stomach pH, whereby pH values of 1.54, 1.58 and 2.01 were recorded 12 h after feeding in O. mossambicus, T. rendalli and C. gariepinus, respectively. Protease and amylase activities were significantly higher (P < 0.05, anova ) in the tilapias than in C. gariepinus. The tilapias may be pre‐adapted to produce more protease and amylase to digest plant material, which is more difficult to digest than animal matter. In all species amylase activity was significantly higher in the proximal intestine than in the other parts of the digestive tract (P < 0.05, anova ). The highest protease activity was recorded in the distal intestines. This is because of the alkaline pH recorded in the proximal and distal intestines, which favours amylase and protease activity, respectively. Lipase activities were significantly higher (P > 0.05) in C. gariepinus than in both tilapias. Marginal cellulase activities were recorded in all species. It is inferred here that phylogeny and not diet may be the main factor influencing enzyme activities, as all fish were fed a similar diet.     

Studies on mammalian glucoamylases with special reference to monkey intestinal glucoamylase

1. Highly purified preparations of glucoamylase were obtained from liver, spleen and intestine of the monkey. The enrichment factor was lower for intestine (60-fold) compared with that of liver (1200-fold) and of spleen (2000-fold) but the final specific activities were of a similar magnitude. 2. The liver and spleen enzymes had maximum activity at pH4.8 whereas the intestinal enzyme showed an optimum at pH5.8. The K(m) values for both starch and maltose with spleen and liver enzymes were higher than for the intestinal enzyme. With the intestinal enzyme, the V(max.) values were higher for both starch and maltose than those of the spleen and liver enzymes. 3. Gel filtration on Sephadex G-200 under identical conditions revealed that liver and spleen enzymes emerge from the columns much later than the intestinal enzyme. 4. Evidence is presented that the glucoamylase activity of the intestinal mucosa is exhibited by the maltase II fraction. 5. Tris, pentaerythritol and turanose inhibited glucoamylase from all the three tissues, but turanose inhibited the spleen and liver enzymes to a higher degree than the intestinal enzyme.     

pH值对中国龙虾消化酶活力的影响

采用酶学分析方法研究了pH对中国龙虾胃蛋白酶、类胰蛋白酶、淀粉酶、纤维素酶和脂肪酶活力的影响。结果表明,在设定的pH范围内,中国龙虾各消化酶的活力均随着pH的升高呈现先升后降的变化趋势。其中,胃、肠、肝胰腺内胃蛋白酶最适pH均为2.2,类胰蛋白酶最适pH分别为8.8-9.2、8.4、8.8,淀粉酶最适pH分别为7.0、7.0、7.4,纤维素酶最适pH分别为4.2、4.2-4.6、5.4,脂肪酶最适pH分别为7.2-7.6、7.2、6.8-7.2。同时测得中国龙虾胃、肠、肝胰腺内的生理pH分别为5.33、6.93、6.60。中国龙虾的消化酶活力存在器官特异性。在最适pH下,胃蛋白酶活力顺序为胃>肠>肝胰腺,类胰蛋白酶、纤维素酶、脂肪酶的活力顺序均为肝胰腺>肠>胃,淀粉酶的活力顺序为肠>肝胰腺>胃。