Seasonal feeding activity and ontogenetic dietary shifts in crucian carp,Carassius carassius

Synopsis A pronounced seasonal cycle in the activity of the intestinal alkaline phosphatase indicated a 6 month pause in feeding by crucian carp in an anoxic pond during winter. Zero enzyme activity was not reached until ice cover of the pond was complete, although even in the preceding two months no food was found in the guts of the fish. The summer diets of four size classes of fish consisted of varying proportions of planktonic and benthic arthropods, including Odonata which were consumed exclusively by the largest size class of crucian carp. Chironomid larvae were consumed even by the smallest fish investigated, although planktonic Cladocera were preferred by this size class. Benthic Cladocera were almost totally absent in the diet of fish> 10 cm. Habitat segregation between size classes developed synchronously with the shift to benthic food. The stunting of crucian carp populations in small ponds is interpreted as resulting from limited resources.     

The thermal properties of intestinal alkaline phosphatase of three kinds of deep-water fish

1. The thermal properties of intestinal alkaline phosphatase were investigated with three species of deep-water fish in the temperature range of 0-70 C. 2. A relationship was found between the thermal stability of the enzyme and the fish origin. 3. Maximum activity of alkaline phosphatase of the fish that originated in tropical water, namely, Aphanopus carbo and Epigonus telescopus was 60 C, whereas the respective maximum enzyme activity of Etmopterus princeps that originated in the burial zone was 30 C. 4. A breakpoint at 10 degrees C in the Arrhenius plot of emzyme activity in the case of A. carbo and a lack of a break point in the case of E. telescopus and E. princeps, are in accordance with the stenothermic nature of the former and the everythermic nature of the two latter fish species.     

Histochemical studies on the effect of thyroid hormone on alkaline and acid phosphatase activities in liver of fish and amphibia.

The Lata fishes (Ophicephalus punctatus) showed increased alkaline and acid phosphatase activities in liver after immersion for 15-30 days in thyroxine-containing medium (0.025 mug/ml). A single injection of thyroxine (1-2 mug/g of body weight) caused increased acid phosphatase activity in liver of Lata fish in comparison to the controls on the 5th day after experiment but the alkaline phosphatase activity remained unchanged. Both alkaline and acid phosphatases showed increased activities in liver of Lata fishes treated with a single injection of 4 mug of thyroxine per g of body weight on the 5th day. Immersion of Lata fishes in thiourea solution (1 mg/ml) for 15 days did not show any alteration in alkaline or acid phosphatase activities but these enzyme activities decreased after 30 days' immersion in thiourea solution in comparison to the controls. A seasonal variation of alkaline and acid phosphatase activities was observed in liver of Lata fishes. More alkaline phosphatase activity was found in liver of summer fishes than in winter fishes. The winter fishes showed more acid phosphatase activity than the summer fishes. Three consecutive injections of thyroxine (0.1 mug/g of body weight) to toads (Bufo melanostictus) caused increased alkaline and acid phosphatase activities in liver on the 5th day of the experiment, in comparison to the controls.     

Effects of hexavalent chromium at non-lethal concentrations on the enzymology of the intestine of Salmo gairdneri and Dicentrarchus labrax (Pisces)

Effects of non lethal concentrations of hexavalent chromium on intestinal enzymology of Salmo gairdneri and Dicentrarchus labrax (Pisces). The effects of an exposure to potassium dichromate on intestinal enzyme activities (Alkaline phosphatase, maltase, leucine amino peptidase and ATPases) have been studied on a fresh water fish (Salmo gairdneri) and a salt water fish (Dicentrarchus labrax). Fish were exposed at seasonal temperatures (13 or 21 degrees C) to toxic concentrations equal to 1/10 of the 24 h-LC 50 (i.e. 18 mg/l Cr for trout and 5 mg/l Cr for bass) during respectively 13 and 21 days. Intoxicated trout stopped feeding and showed a decrease in their intestinal weight at the end of the experiments. A decrease of brush border membrane activities (Alkaline phosphatase, maltase and leucine amino peptidase) were also observed. These alterations have been interpreted as the consequence of the chromium induces fasting. Intoxicated bass showed no alterations of their feeding habits. Two specific effects of chromium on enzyme activities have been found: a severe decrease of the alkaline phosphatase activity and an increase of the Na/K ATPase activity. These enzyme activities could be useful indicators of chromium intoxication in marine fish.     

Characterization of Digestive Enzymes in the Intestine of the Pacific Halibut Hippoglossus stenolepis Schmidt, 1904 and the Starry Flounder Platichthys stellatus (Pallas, 1788)

The activity and temperature characteristics of hydrolases (maltase, α-amylase, total proteinase, alkaline phosphatase) in the intestine of Far Eastern flatfishes were studied. The character of the temperature dependence differed among the investigated enzymes. The activity of individual hydrolases in the two fish species varied. The results of this study suggest that flatfish intestinal hydrolases are efficient at low temperatures and that the digestive process in flatfishes is well adapted to the conditions in which it functions, thus ensuring optimal vital activity at low temperatures in the Far Eastern seas.Original Russian Text Copyright © 2005 by Biologiya Morya, Korostelev, Nevalenny, Levchenko.     

Temperature dependent characteristics of intestinal glycyl-L-leucine dipeptidase in boreal zone fish

Three kinds of boreal zone fish were investigated for gastrointestinal glycyl-L-leucine (GL) dipeptide cleaving activity as a function of feeding stage and seasonal changes. The enzyme activity tested in the perch (Perca fluviatilis L.) intestine increased steadily during digestion and rapidly disappeared after completion. The temperature characteristics and the seasonal changes in dipeptide cleaving activity in pike perch (Stizostedion lucioperca L.) and bream (Abramis brama L.) were studied. In summer, the maximal activities in the pike perch and the bream were found at temperatures of 40 and 30 degrees C, respectively. In winter, the temperature of maximal activity in pike perch fell to only 30 degrees C, whereas no changes were observed in bream. The activation energies in bream and pike perch were several times lower in winter than in summer. Seasonal changes in the dipeptide cleaving activity at low temperature relative to that at the temperature of maximal activity were found. At high temperatures, the stability of the enzyme decreases in winter and increases in summer, but in the presence of a substrate the thermal stability of the enzyme increases both in winter and in summer. In our experiments, we found that in these fish, GL dipeptidase was unstable at 0 and -10 degrees C.     

Role of alkaline phosphatase in phosphate uptake into brush border membrane vesicles from human intestinal mucosa

In order to elucidate the physiological function of intestinal alkaline phosphatase, the characteristics of human intestinal alkaline phosphatase bound to brush border membrane vesicles were compared under optimal and physiological pHs. The Km value of this enzyme towards p-nitrophenylphosphate at the physiological pH was lower than that at the optimal pH. At the physiological pH, phosphate, arsenate and vanadate competitively inhibited the alkaline phosphatase activity, as they did at optimal pH, and the K1 values of these inhibitors at the physiological pH were also lower than those at the optimal pH. The effects of various inhibitors and antibody to human intestinal alkaline phosphatase on phosphate uptake into brush border membrane vesicles were investigated. The results indicated that phosphate uptake was affected by various inhibitors and the antibody to human intestinal alkaline phosphatase, but L-homoarginine, levamisole, and ouabain had no effect. From the above findings, it is strongly suggested that human intestinal alkaline phosphatase may function as a phosphate binding protein at low phosphate concentrations under physiological conditions.     

Modulation of alkaline phosphatases in LoVo, a human colon carcinoma cell line

LoVo, a continuous cell line derived from a human colon carcinoma produces two alkaline phosphatases: the heat-labile, L-homoarginine-insensitive, intestinal form, characteristic of its tissue of origin and the heat-stable, term-placental form, ectopically produced by a variety of tumors. Under basal conditions the activity levels of both enzymes are similar. Hyperosmolality and sodium butyrate induce increased levels of activity of the two alkaline phosphatases in a disparate fashion; whereas hyperosmolality augments the activity of both to the same extent, the effect of butyrate is more pronounced on the activity of the intestinal enzyme. When the two inducers are combined, induction of term-placental alkaline phosphatase is additive and that of the intestinal enzyme is synergistic. The effect of hyperosmolality is blocked by cycloheximide, and induction by sodium butyrate is inhibited by thymidine, cordycepin and cycloheximide. The known alkaline phosphatase inducer, prednisolone, has no effect on the enzymes of LoVo cells. Our results suggest that in these tumor cells the activity levels of the closely homologous term-placental and intestinal alkaline phosphatases appear to be independently controlled.     

The response of alkaline phosphatase to osmoregulatory changes in the trout,Salmo gairdneri

Summary The relationship between alkaline phosphatase and environmental salinity was examined in the rainbow trout and the migratory rainbow (steelhead),Salmo gairdneri. The enzyme activity in tissues involved in osmoregulation was strongly correlated with the adaptation salinity and thus to the degree of salt and fluid transport in those tissues. After transfer from freshwater to seawater, the specific activity of the enzyme increased over 260% in the intestine, decreased by 50% in kidney, and was unchanged in the liver, an organ not directly involved in osmoregulation. The sea-run steelhead trout response was similar to the nonmigratory rainbow; although, the pre-migratory transformation (smoltification) had no effect on enzyme activity. Amino acid inhibitors of alkaline phosphatase significantly reduced fluid absorption in the isolated intestine of rainbow trout, reaffirming the relationship between the enzyme and fluid movement. Electrophoretic identification of trout alkaline phosphatase isozymes, clearly distinguishes the enzyme from different tissue origins. However, from the analysis of intestinal electrophoretic patterns, osmoregulatory adjustments are not associated with the induction of new alkaline phosphatase isozymes, or in the large scale preferential stimulation of one of the two existing intestinal isozymes over the other.     

Effects of thermal acclimation on the relaxation system of crucian carp white myotomal muscle.

Many cyprinid fish are able to compensate for a decrease in ambient temperature by process of physiological adaptation in the function of muscles. In the winter habitat of crucian carp (Carassius carassius L.), low temperature is associated with simultaneous oxygen shortage. Because of the oxygen deprivation, there is probably little space for compensatory adaptation because positive thermal compensation would increase energy demand and accelerate depletion of glycogen reserves. Thus, we assumed that the crucian carp, unlike many other cyprinid fish, would not show positive thermal compensation but either no compensation or inverse compensation in muscle function. To test this hypothesis in the relaxation system of skeletal muscles, we determined the parvalbumin content and the activity of sarcoplasmic reticular (SR) Ca-ATPase in white myotomal muscle of winter- and summer-acclimated crucian carp. In the laboratory, the winter fish were kept at 2 degrees C and the summer fish at 22 degrees C for a minimum of 3 weeks before the experiments. The specific activity of SR Ca-ATPase at low experimental temperature (2 degrees C) was similar in summer- and winter-acclimated fish (0.26 +/- 0.04 vs. 0.25 +/- 0.04 mM/mg/min; P > 0.05). Because of the bigger Q(10) of cold-acclimated carp, the enzyme activity at 30 degrees C was higher in cold-acclimated winter fish than in warm-acclimated summer fish (7.42 +/- 0.90 vs. 5.18 +/- 0.53 mM/mg/min; P < 0.05). In contrast, the yield of SR protein was 70% higher in summer than winter fish (0.315 +/- 0.045 vs. 0.187 +/- 0.017 mg/g; P < 0.001). Because of these opposing changes, total Ca-ATPase activity of SR (per gram muscle weight) remained relatively constant. Similarly, the parvalbumin content of the myotomal muscle was not different between summer (4.09 +/- 0.95 mg/g) and winter (3.70 +/- 0.60 mg/g) fish. Although there were no seasonal changes in the total relaxing system of the crucian carp white myotomal muscle, the same activity of SR Ca-ATPase in winter fish was obtained with less amount of SR pump protein, owing to the increased catalytic activity of the enzyme. The higher catalytic activity of winter fish SR Ca-ATPase might be caused by differences in fatty acid composition noted in membrane lipids; i.e., fewer saturated fatty acids and more n-6 polyunsaturated fatty acids (PUFAs), at the expense of n-3 PUFAs, were present in the SR of cold-acclimated winter fish. Temperature-induced changes in enzyme protein, however, cannot be excluded. Thus, the present results indicate the absence of positive thermal compensation in the relaxing system of crucian carp white muscle. It seems, however, that lipid composition of SR membranes and temperature dependence of SR Ca-ATPase are altered by seasonal acclimation.     

Phosphate binding in the active site of alkaline phosphatase and the interactions of 2-nitrosoacetophenone with alkaline phosphatase-induced small structural changes

To monitor structural changes during the binding of Pi to the active site of mammalian alkaline phosphatase in water medium, reaction-induced infrared spectroscopy was used. The interaction of Pi with alkaline phosphatase was triggered by a photorelease of ATP from the inactive P(3)-[1-(2-nitrophenyl)]ethyl ester of ATP. After photorelease, ATP was sequentially hydrolyzed by alkaline phosphatase giving rise to adenosine and three Pi. Although a phosphodiesterase activity was detected prior the photorelease of ATP, it was possible to monitor the structural effects induced by Pi binding to alkaline phosphatase. Interactions of Pi with alkaline phosphatase were evidenced by weak infrared changes around 1631 and at 1639 cm(-1), suggesting a small distortion of peptide carbonyl backbone. This result indicates that the motion required for the formation of the enzyme-phosphate complex is minimal on the part of alkaline phosphatase, consistent with alkaline phosphatase being an almost perfect enzyme. Photoproduct 2-nitrosoacetophenone may bind to alkaline phosphatase in a site other than the active site of bovine intestinal alkaline phosphatase and than the uncompetitive binding site of L-Phe in bovine intestinal alkaline phosphatase, affecting one-two amino acid residues.     

myo-inositol action on gerbil intestine: alterations in alkaline phosphatase activity upon phosphatidylinositol depletion and repletion in vivo

The influence of phosphatidylinositol (PI) on intestinal alkaline phosphatase activity was studied in myo-inositol deficient gerbils. A reduction of membrane PI in intestinal mucosa to 30-40% of the control was produced by feeding female gerbils a myo-inositol-deficient diet containing coconut oil for 2 weeks. As expected, the animals developed typical intestinal lipodystrophy with abnormal fat accumulation. In the PI-depleted animal, intestinal alkaline phosphatase activity was reduced to 20-30% of the control group. The levels of both membranous and soluble enzymes in intestinal mucosa were affected, but there were no changes in liver, kidney and plasma levels. When the lipodystrophic gerbils were given dietary myo-inositol, the complete repletion of intestinal membrane PI to the control level occurred 36 h later, whereas membrane-bound alkaline phosphatase activity in intestine was not restored to the control level until 72 h later. Administration of cycloheximide or actinomycin D did not block this enzyme induction. Lymphatic output of triacylglycerol into the bloodstream was stimulated 10-fold at 18 h of myo-inositol repletion, but there was no parallel increase in the activity of alkaline phosphatase in plasma during this early phase of intestinal recovery. Thus, these data suggest a possible regulatory role of PI in the processing and/or turnover of alkaline phosphatase in vivo, but a negative role of alkaline phosphatase in lipid transport across gerbil intestine.     

Prenatal diagnosis of cystic fibrosis by microvillar enzyme assay on a sequence of 258 pregnancies

Summary Prenatal diagnosis of cystic fibrosis by microvillar enzyme assay on amniotic fluid supernatant has been carried out on 258 sequential pregnancies with a 1 in 4 recurrence risk, all with known outcome. In general the three enzymes evaluated, -glutamyltranspeptidase, aminopeptidase M and the intestinal isoenzyme of alkaline phosphatase, showed a high degree of concordance. However, there were two unusual patterns of microvillar enzyme activity; in seven cases a low -glutamyltranspeptidase activity was associated with elevated values of intestinal alkaline phosphatase, and in ten cases there were isolated low values of intestinal alkaline phosphatase. The former pattern was found to be associated with cystic fibrosis in five cases, while the latter was associated with a normal outcome in all ten cases. A retrospective analysis of enzyme values suggested that the optimal system for minimizing false positives and false negatives was to define foetal cystic fibrosis as a sample where two of the three microvillar enzymes were below a cut-off of half the median value for the gestational week. If such scoring were applied to the cases where conventional microvillar enzyme patterns were observed, the false positive rate was 2.3% and the false negative rate 4.4% between 17 and 20 weeks of gestation.     

Alkaline phosphatase isozymes of Xenopus laevis embryos and tissues.

Alkaline phosphatase was obtained by treating embryos of Xenopus laevis with n-butanol at different developmental stages from gastrula to tadpole; the enzyme was also obtained from adult kidney, liver, and intestinal mucosa. Purification was carried out by gel filtration and polyacrylamide gel electrophoresis. The enzyme activity is chromatographically spearated into two peaks, with molecular weights of approximately 200,000 and 400,000. Alternatively, two groups may be characterized on the basis of their electrophoretic mobilities, which correspond to the different molecular weight classes. Effects of pH, temperature, inhibitors, and substrate concentration were studied. The kinetic and physical properties of the two alkaline phosphatase isozymes are similar, and are comparable to the properties reported for this enzyme from other vertebrates. Alkaline phosphatase activity increased sharply at the gastrula stage and reached a plateau at the late tailbud stage. During this period there was an 18-fold increase in activity.     

Purification and characterization of phytase from rat intestinal mucosa.

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8 or 3.1.3.26) was purified from rat intestinal mucosa. The purified enzyme preparation exhibited two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular masses of 70 kDa and 90 kDa. Rabbit antisera prepared against the 90K subunit cross-reacted with the 70K subunit on immunoblotting. The peptide maps of the 70K and 90K subunits were similar, and the N-terminal amino acid sequences of the two subunit proteins were almost identical. Treatments to remove sugar moieties from the proteins showed that the two subunit proteins had different oligosaccharide chains, although the difference in their molecular masses was not due to the difference in their oligosaccharide compositions. The purified enzyme also showed activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1), but the properties of the two enzyme activities were different; the optimum pH for phytase activity was 7.5, while that for alkaline phosphatase was 10.4. Phytase activity did not necessarily require divalent cations, while Mg2+ was essential for alkaline phosphatase activity. Phenylalanine, a specific inhibitor of intestine-type alkaline phosphatase had no effect on the phytase activity.     

Sub-lethal effect of synthetic pyrethroid pesticide on metabolic enzymes and protein profile of non-target Zebra fish,Danio rerio

Extensive application of pesticide in agricultural field affects the enzymatic activity of non-target animals, including fishes. In this study, the impact of sublethal concentration of fenvalerate on marker enzymes of freshwater Zebra fish was evaluated. Pesticide-induced stress can specifically affect non target fishes, through elevated level of reactive oxygen species which is responsible for biochemical, cell metabolism and physiological activities. The oxidative stress mediated by fenvalerate at sub lethal concentrations after 28 days of exposure of Zebra fish. Following 28 days of exposure of pesticide, catalase, superoxide dismutase, aspartate amino transferases, alanine amino transferase, alkaline phosphatase and acid phosphatase were assessed. Results revealed reduction of superoxide dismutase activity after 28 days of exposure in sub lethal concentration of fenvalerate in liver and gills. In liver, catalase activity was found to be less in fenvalerate exposed fish than control fish. In liver, increase of 75.75% aspartate amino transferase and 38% increase in alanine amino transferase in gills. SGPT activity was relatively higher than SGOT suggests more contribution of phyruvalate than oxaloacetate formation. Fenvalerate induced changes in acid phosphatase and alkaline phosphatase activity in the liver and gills of Zebra fish after four weeks of exposure. Fenvalerate induced expression of various stress proteins in gill, liver, followed by muscle. Some proteins lost its intensity due to fenvalerate toxicity. Result revealed that enzyme assays and SDS-PAGE analysis for protein subunits determination is relevant tool to monitor stress in freshwater ecosystem. The findings suggest that in monitoring fenvalerate toxicity programme, enzyme activities can be potent diagnostic tool for fenvalerate induced toxicity.     

Quantitative histochemical study of alkaline phosphatase isoenzymes in the uteri of ovariectomized golden hamsters during estrogenization

The activity of two isoenzymes of alkaline phosphatase was studied in the uteri of ovariectomized golden hamsters. Animals belonging to different groups were daily injected with 10 micrograms of estrogen (octoestrol) once, for 4 and 16 days. The estrogen did not affect the overall activity lf alkaline phosphatase in the uterine luminal epithelium, but decreased the enzyme activity in stroma. Moreover, it was found that prolonged estrogen treatment increases significantly the proportion of alkaline phosphatase isoenzyme of the intestinal type in the uterine luminal epithelium.     

Induction of enhanced alkaline phosphatase activity in the melano-macrophage centres of Oreochromis aureus (Steindachner) through starvation and vaccination

Employing histochemical methods, alkaline phosphatase activity was studied in the melano-macrophage centres of the spleen of the cichlid fish Oreochromis aureus (Steindachner). Enzyme activity was observed to be very low in normal fish. Prolonged starvation induced an enhanced enzyme response. Starvation followed by antigenic stimulation through an intraperitoneal injection of a bacterial vaccine further elevated the levels of alkaline phosphatase activity. The marked response of the pigment-bearing macrophages to bacterial antigen provides further evidence of the lymphoreticular nature of these pigmented cell aggregates. The association of alkaline phosphatase activity and lipofuscin (the most common pigment in fish melano-macrophage centres) with phagocytic cells has been documented in higher animals including     

Isolation and properties of fecal proteins and fecal alkaline phosphatase from germfree and conventional rats.

Fecal proteins from germfree and conventional rats were isolated. The proteins from the two kinds of feces differed in molecular weight, judging from Sephadex gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The conventional feces contained a greater amount of high-molecular-weight and a lesser amount of low-molecular-weight proteins than did the germfree feces. The fecal proteins of both kinds contained carbohydrates. Both feces contained considerable enzyme activity. The germfree feces contained extremely high activity in alkaline phosphatase and leucine aminopeptidase. Both feces showed the same level of trehalase activity. The conventional feces contained higher levels of activity of protease and acid phosphatase than did the germfree feces. Lactase activity was observed only in the conventional feces. The fecal alkaline phosphatase resembled the intestinal enzyme in response to L-phenylalanine inhibition and urea denaturation. From these results it was inferred that the germfree feces contained some of the intestinal proteins and that the conventional feces contained bacterial proteins in addition to intestinal proteins.     

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.