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.     

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).     

Effect of temperature on proteinase activities in intestinal chyme and mucosa of fish of different ecological groups

Effect of temperature on activities of proteinases in intestinal chyme and mucosa was studied in three fish species (pike-perch, zope, roach) belonging to different ecological groups by their nutrition type. There was revealed a significant difference of dependence of enzyme activities in chyme on temperature in the benthophage, roach (a higher level of relative activity in the range of lower temperatures and a wider zone of temperature optimum) as well as of values of apparent energy of activation of the protein hydrolysis process as compared with that in planktoand ichtyophages, zope and pike-perch, which indicates a significant effect of the enteral microbiota proteinases and of nutrition objects on characteristics of hydrolases functioning in fish intestine.     

Effect of temperature on proteinase activities in the intestinal chyme and the mucosa of various ecological fish groups

Effect of temperature on activities of proteinases in intestinal chyme and mucosa was studied in three fish species (pike-perch, zope, roach) belonging to different ecological groups by their type of feeding. There was revealed a significant difference of dependence of enzyme activities in chyme on temperature in the benthophage roach (a higher level of relative activity in the zone of lower temperatures and a larger zone of temperature optimum) as well as of values of apparent energy of activation of the protein hydrolysis process as compared with that in plankto- and ichthyophages--zope and pike-perch, which indicates a significant effect of the enteral microbiota proteinases and of nutrition objects on characteristics of hydrolases functioning in fish intestine.     

Effect of thyroid hormones on activity dynamics of enzymes of intestinal mucosa of juvenile roach <Emphasis Type="Italic">Rutilus rutilus</Emphasis>

The effect of thyroid hormones on activity dynamics of enzymes (proteinases and glycosidases) of intestinal mucosa of juvenile roach Rutilius rutilus was investigated. Application of substances increasing and decreasing the level of thyroid hormones in blood plasma significantly influences the growth rate and the activity of proteinases and glycosidases functioning in the intestinal mucosa. In most cases, the activity level of trypsin-like proteinases and the activity of glycosidases in the fish exposed to triiodothyronine were significantly higher than in the control. The activity level of chymotrypsin-like proteinases in fish form the group with exposure of exogenous triiodothyronine only in the end of the experiment surpassed the values of this parameter in the control fish. In the fish developing at deficiency of thyroid hormones, the growth rate and proteinases activity were significantly lower in comparison with the control.     

Proteinases of kilka <Emphasis Type="Italic">Clupeonella cultriventris</Emphasis> of the Rybinsk Reservoir

The effect of temperature on activity of proteinases of the stomach mucosa, intestinal mucosa, chime, and the entire body of kilka Cluperonella cultriventris is described. Significant differences are revealed in temperature characteristics (t ^o-function, Q ₁₀, and E _act) of the investigated enzymes. The temperature has a larger influence on the activity of intestinal proteinases than the activity of proteinases of the stomach, chime, and the whole kilka organism. The lowest value of E _act within the temperature range of life of this species is characteristic of chime proteinases. Hemoglobinlytic proteinases of various tissues of kilka that substantiate, at pH 3.0 and 5.0 (the action zone of cathespsins), the processes of induced autolysis contribute to efficient alimentation of other fish species. The assumption is made that a high activity level of proteinases of the digestive tract, comparable with that of other fish species of the upper Volga, enables kilka that have assimilated to the Rybinsk Reservoir to feed efficiently within the temperature range characteristic of the water-bodies of this zone.     

The effect of temperature,pH, and heavy metals (copper,zinc) upon proteinase activities in the potential preys for typical and facultative piscivores

Separate and combined action of temperature, pH, and heavy metals (copper and zinc) upon whole body activities of casein-and hemoglobinlytic proteinases were studied in perch (Perca fluviatilis), ruff (Gymnocephalus cernuus), roach (Rutilus rutilus), kilka (Clupeonella cultriventris), and common carp (Cyprinus carpio). Also, these activities were determined in cyprinids’ underyearlings (in sum) dominating the food of typical and facultative piscivores. It was revealed that, following a decrease in temperature to 0°C and in the presence of heavy metals (copper and zinc) at concentrations of 10 mg/l, casein-and hemoglobinlytic proteinase activities decreased statistically significantly in all studied fish. Both decrease and increase in pH values lead to less pronounced changes in the enzymatic activities varied in different species. As a rule, decrease in pH values resulted in considerable increase of the hemoglobinlytic activity, while pH increase caused inconsiderable enzyme activation. Combined action of natural factors and, in some cases, combined action of low temperature and metals, changed the caseinlytic activity to a greater extent than at their separate actions. Magnitude of multifactor effects in most cases is lower than of bifactor effects of temperature and metals.     

Effects of Temperature and pH on Activity of Proteinases of Intestinal Mucosa and Enteral Microbiota of Fish

Separate and combined effects of temperature and pH on activity of proteinases of intestinal mucosa and enteral microbiota were studied in several fish species. There were revealed peculiarities of temperature function of enzymes in microbiota of ichthyophages (a high level of relative activity in the whole range of their vital activity), which indicate the presence of victim-symbiotic adaptations in fish feeding in winter.     

The influence of testosterone on activity of glycosidases and proteinases in the intestine of the sterlet (Acipenser ruthenus)

Observation of the influence of testosterone (0.7 mg/kg) on the activity of glycosidases and proteinases, which function in the chyme and the mucosa of the sterlet intestine (Acipenser ruthenus), revealed a decrease in enzyme activity of both chains as against that one typical for intact individuals. The activity of the investigated enzymes changes in both the experimental and control groups of fish. Nevertheless, they have unlike dynamics of this activity. In a number of cases, we indicated a significant increase in enzyme activity of both chains in comparison to the control group. We also observed an increase in activity of chyme glycosidases, which was significant on the 21st day as compared to intact fish.     

Effect of dexamethasone on activity of glycosidases and proreinases of intestine of sterlet <Emphasis Type="Italic">Acipenser ruthenus</Emphasis>

Effect of dexamethasone (2.0 mg/kg) on activity of enzymes hydrolyzing proteinaceous and carbohydrate food components in the intestine of the starlet Acipenser ruthenus is studied. Dexamethasone modifies the activity of proteinases more than of glycosidases. As a rule, the hormone significantly decreases the level of proteolytic and general amylolytic activity of the intestinal mucosa and chyme in comparison with intact fish on the first day into the experiment and increases it on the 7th or 14th day. The dynamics of activity was different in enzymes of different chains (glycosidase and proteinase) and preparations (the mucosa and chyme).     

Influence of biogenic metals (Cu,Zn) on the activity of carbohydrases in juvenile fish in vitro

It was revealed that intestine and whole-body amylolytic activity (AA) in juvenile fish serving as potential feeding items for piscivores significantly decreases in the presence of Cu and Zn ions within a wide range (0.1–25 mg/l) of concentrations in vitro. In most of the studied fish species, Cu and Zn decrease the activities of carbohydrases in the intestine mucosa stronger than in the whole-body. On the other hand, in piscivore-facultative benthivore perch, which have the least AA, the inhibiting effect of Cu and Zn ions is 1.5 to 2 times higher in the whole body than in the intestine. The study data suggest that Cu and Zn ions at concentrations found in organisms that serve as fish food may not only reduce the rate of initial stages of carbohydrates hydrolysis in the juvenile fish intestine, but also considerably decrease the potential contribution of the food objects to the digestion processes in typical and facultative piscivores.     

Effect of temperature on proteinase activities of enteral microbiota and intestinal mucosa of fish of different ecological group

Effect of temperature on proteinases activities of enteral microbiota and of intestinal mucosa was studied in five fish species (roach Rutilus rutilus, crucian carp Carassius carassius, common perch Perca fluviatilis, pike-perch Zander lucioperca, and pike Esox lucius) belonging by the nutrition type to different ecological groups. Essential differences of temperature characteristics of proteinases of intestinal mucosa and of enteral microbiota are revealed in fish belonging by the nutrition type to different ecologic groups. The character of the t0-function of proteinases of intestinal mucosa and enteral microbiota by casein and hemoglobin as a rule is different. The highest values of relative proteinases activities for casein in the zone of low temperatures (38 and 45.3 % of the maximal activity) are found at study of proteinases of enteral microbiota in common perch and crucian carp. The latter indicates a significant adaptability of the enteral microbiota proteinases of common perch and crucial carp to functioning at low temperatures.     

Effect of temperature on proteinase activites of enteral microbiota and intestinal mucosa of fish of different ecological groups

Effect of temperature on proteinases activities of enteral microbiota and of intestinal mucosa was studied in five fish species (roach Rutilus rutilus, crucian carp Carassius carassius, common perch Perca fluviatilis, pike-perch Zander lucioperca, and pike Esox lucius) belonging by the nutrition type to different ecological groups. Essential differences of temperature characteristics of proteinases of intestinal mucosa and of enteral microbiota are revealed in fish belonging to different ecological groups. The character of the t-function of proteinases of intestinal mucosa and enteral microbiota for casein and hemoglobin as a rule is different. The values of the apparent E _act proteinases of intestinal mucosa for casein in most cases are higher than those of enteral microbiota, while those for hemoglobin, on the contrary, are lower. The lowest values of relative proteinase activities for casein in the zone of low temperatures (38 and 45.3% of the maximal activity) and the E_act value (less than 2.0 kcal/mol) are found at study of proteinases of enteral microbiota in common perch and crucian carp. The latter indicates a significant adaptability of the enteral microbiota proteinases of common perch and crucian carp to functioning at low temperatures.     

Effect of pH on the activity of proteinases in intestinal mucosa,chyme, and microbiota of fish from the Cuciurgan reservoir

Proteolytic activities of the intestinal mucosa, chyme, and enteral microbiota have been studied in a wide range of pH values in five fish species from the Cuciurgan reservoir (Moldova). Differences in pH dependence of the intestinal proteinase activity of fish are determined by their feeding type. The maximum activity of proteinases is found in the pumpkinseed Lepomis gibbosus. The minimum activity of proteinases has been demonstrated by the zander Zander lucioperca. The pH optimum of the mucosa and chyme in all fish species (except for the European perch Perca fluviatilis) is 10. The pH optimum of the intestinal microflora varies from 6.0 (in the common carp Cyprinus carpio) to 10 (in the crucian carp Carassius carassius), whereas that in the perch from the Cuciurgan and Rybinsk reservoirs is 7. The majority of fish species, mostly Zander lucioperca and Lepomis gibbosus, are characterized by high proteniase activity of the microbiota, in the pH ranging from 6 to 9. It is assumed that proteinases in the enteral microbiota of fish are able to make up for the relatively low activity of those synthesized by their digestive system in the range of low pH values.     

Enzyme-forming function of digestive glands after administration of polypeptide C, copepsidyl and panintestine

Polypeptide C (molecular weight 2640 Dalton) extracted from artificial gastric juice), copepsidyl (containing high dosage of pepsin) and panintestine (polyenzyme drug) are studied for their effect on the activity of digestive enzymes of glandular gastric element pancreas and small intestine of rats. It is established that all mentioned drugs stimulate enzymogenesis in the analyzed organs. The activity of pepsin increases in homogenates of gastric mucosa, the activity of trypsin, total proteinases, carboxypeptidase and amylase grows in pancreas homogenates, and that of leucineaminopeptidase--in small intestine.     

Serine proteinases of lower vertebrates

Recent data on the effect of serine proteinases of lower vertebrates are generalized. Hydrolysis specificity and kinetics of different synthetic substrates, dependence of the activity of enzymes on pH, their irreversible inhibition by chloromethyl ketones of amino acids and peptides as well as high-molecular proteinase inhibitors are considered in detail. The data testify to the fact that chymotrypsins and trypsins of higher vertebrates and serine proteinases of lower vertebrates act as an acid-base catalysis. Enzymes in the pyloric cacca of fishes are in the state of proenzymes and are transformed into an active form with the aid of their own proteolytic factors. The esterase and proteolytic activity of fish proteinases is concentrated in the same active site and reaches the highest values at pH 7,8. New data are presented on particularities of the lower vertebrate proteinases, on the similarity and differences in their specificity. A distinct difference is shown in the nature of the binding site of the active centre in a number of serine proteinases of fishes as compared to chymotrypsin and trypsin of higher vertebrates.     

Proteinase,Lipase and Amylase Activities in the Small Intestine of Pigs Suffering from Colienterotoxaemia

The activities of proteinases, lipases, amylases and the activities of proteinase inhibitors, as well as the numbers of Escherichia coli in the contents from the small intestine were examined for pigs suffering from colienterotoxaemia and for healthy pigs. Enzyme activities were determined using an agar diffusion test. Compared with healthy animals the activities of proteinases and amylases in diseased animals were reduced while lipases showed increased activity. In pathologically changed contents showing large numbers of E. coli, proteinases could not be demonstrated; however, proteinase inhibitors were found in these contents. In healthy animals, proteinase inhibitors were not demonstrated in ingesta-con-taining contents. In diseased animals, E. coli were found in large numbers in all parts of the small intestine. In healthy animals, E. coli was demonstrated especially in the posterior part of the small intestine and regularly in small numbers. The possible influence of digestive enzymes, especially proteinases and their inhibitors, on enterotoxins from E. coli is discussed.     

小白鼠发育过程中胃和大肠蛋白酶活性的变化

采用活性电泳方法对小白鼠发育过程中胃和大肠蛋白酶活尾进行了研究。结果发现：（１）出生前、胃和大肠蛋白酶种类少、活性弱,出生后,胃和大肠蛋白酶的种类多,活性增加;（２）胃肠蛋白酶可能与其结构的构建和功能建立有密切关系;（３）食物 刺激可能对胃肠蛋白酶有显著影响。     

Impact of polychlorinated biphenyls on the activity of intestinal proteinases and carbohydrases in juvenile roach Rutilus rutilus (L.)

The chronic effects that polychlorinated biphenyls (PCBs) have on the activities of proteinases and carbohydrates in intestinal mucosa and chyme in juvenile roach Rutilus rutilus (L.) have been studied for the first time. Upon consuming food with PCB 50.8 ng/g wet weight for 218 days, the exposition of fingerlings in aquariums with dirt bottoms (contents PCB 425.6 ng/g dry weight) reduces the proteolytic activity of mucosa to a greater degree than amylolytic activity; the activity of sucrase changes in different directions. In 2-year-old fish receiving PCB only with food (50.8 ng/g wet weight bottom), proteolytic and amylolytic activities of the mucosa are reduced 18 and 35% in comparison with the control (52 days); the sucrose activity is reduced 13% (52 days) and 22% (169 days). In most cases the relation of amylolytic activity to proteolytic activity in fish of the experimental groups is less than in the control. The enzyme activities in chyme change in different directions, depending on the terms and conditions of the experiment.     

The characteristics of protein hydrolysis on the digestive-transport surfaces of the cestode Eubothrium rugosum and of the intestines of its host--the burbot]

The functioning of different proteinases hydrolysing proteins in a wide pH range, most of which display activity in the alkaline zone of pH, on the digestive-absorptive surfaces of the parasite and host has been investigated. The dynamics of desorption of these proteinases from the intestine of fishes and tegument of cestodes has been studied. It has been shown that the worms possess less proteolytic activity and less capacity for adsorption of proteinases as compared to the intestines of their hosts. The dependence of proteolytic activity of desorbed fractions on the incubation medium temperature has been noted: with the increase in temperature the enzymes, bound closely with the membranes, increase their capacity to hydrolyse proteins. The predominance in cestodes, as compared to the intestine, of easily desorbed fractions D1 and D2 (in the percent ratio of the total proteolytic activity of all fractions) has been detected.