Branched-chain amino acid aminotransferase activity in the tissues of lake trout

The enzyme branched-chain amino acid aminotransferase (BCAT) was found in five tissues of fingerling lake trout, Salvelinus namaycush, (listed in order of decreasing tissue specific activity): posterior kidney, skeletal muscle, gill, liver, and anterior kidney. This pattern is consistent with that found in other animals. The results of this study seem to indicate that BCAT in the liver of lake trout has a higher specific activity than that of the rat and that the specific activity is higher in both the liver and skeletal muscle than it is in these organs of the chick.     

Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog

Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min^?1 mg^?1 at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.     

Purification and subunit structure of mouse liver cystathionase

Cystathionase has been purified from mouse liver by ammonium sulfate precipitation, ethanol precipitation, column chromatography on DEAE-cellulose and on hydrox-ylapatite, as well as Sephadex G-200 gel filtration. These procedures yielded a chromatographically homogeneous enzyme which was purified more than 1000-fold relative to whole liver extract. Overall recovery was approximately 4%. The purified enzyme does not contain detectable carbohydrate and migrates as a single protein component on analytical disc gel electrophoresis. A sedimentation coefficient of 8.3 S has been determined for the active enzyme by rate zonal centrifugation in glycerol gradients. This value suggests a molecular weight for the native enzyme of approximately 160,000 g/mol, a value similar to that estimated by gel filtration. Following sodium dodecyl sulfate gel electrophoresis in the presence of reducing agent and at different gel concentrations, a single protein component with a molecular weight of 40,000 g/mol was obtained. Thus, the enzyme appears to consist of four subunits of equal size. The K_m value for cystathionine at pH 8.1, 37 °C, and in the presence of 1 mm dithioerythritol is approximately 1 mm.     

A heparan sulfate-degrading endoglycosidase from rat liver tissue

Incubation of a rat liver lysosomal fraction with [³⁵S]heparan sulfate resulted in degradation of the polymer to oligosaccharides, demonstrating the presence of a heparan sulfate-degrading endoglycosidase. Judging from the size of the oligosaccharides, representing degradation end-products, only a limited number of the glycosidic linkages in the heparan sulfate molecule would seem to be susceptible to the heparitinase.The pH-dependence of the enzyme (active at pH 5.6; inactive at pH 3.8) was found to differ from that of liver hyaluronidase (active at pH 3.8; inactive at pH 5.6), suggesting that the heparitinase is a previously unknown enzyme.     

Fish-Community Response To Protective Liming in Thrush Lake, Minnesota

A protective limestone treatment was applied to an acid-sensitive lake in northeastern Minnesota as part of the Acid Precipitation Mitigation Program. This 6–year study evaluated the impact of that treatment on lakes in the upper Midwest that experience episodes of acid stress but have not lost basic species integrity and community structure. Several changes in the fish community can be directly or indirectly attributed to the addition of 4.6 tonnes of calcium carbonate early in the third year of the study. An almost 30–fold increase in the population of Pimephales promelas(fathead minnow) a year after liming, based on mark-recapture estimates from trap netting and snorkeling, was attributed to a pH increase and a three-fold increase in the calcium concentration of the epilimnion. After the initial increase, the abundance of fathead minnows declined in subsequent years, as did the elevated pH and calcium concentrations. The Salvelimis fontinalis(brook trout) population also increased in the lake following application of limestone, but this was due in part to closing the lake to fishing. An increase in survival of stocked brook trout to age 1+ and an increase in growth of older brook trout after liming were attributed to the increased forage that the fathead minnows provided. Fathead minnows may have also reduced predation pressure on young brook trout by older brook trout. This study demonstrated that liming of a slightly acidic lake did not adversely affect the integrity of the fish community, and in fact may have increased the abundance and biomass of the forage fish community and indirectly increased the survival, abundance, and growth of brook trout.     

Concentrations and distribution of Fe,Zn and Cu in tissues of the white sucker (Catostomus commersoni) in relation to elevated levels of metals and low pH

This study examines whether the process of lake acidification influences the accumulation of Fe, Zn and Cu in the tissues of the white sucker (Catostomus commersoni). Concentrations of Fe, Zn and Cu were measured in the liver, kidney and muscle of white sucker sampled from 4 acidic (pH range 4.8–5.3), 1 slightly acidic (pH = 5.8) and 3 circumneutral (pH = 6.3, 6.4) lakes located in south-central Ontario, Canada. Pearson product-moment correlation coefficients were used to determine relationships between average elemental concentrations in the 3 tissues and both sediment and water metal concentrations plus lake pH, DOC and alkalinity. Despite the 1000-fold difference in H+ concentration among the 8 study lakes, tissue concentrations of Fe, Zn and Cu did not correlate with lake pH. Average Fe, Zn and Cu tissue concentrations did not correlate with metal concentrations in lake water. Only Zn concentrations in the liver and muscle were correlated with Zn concentrations in the sediment (r = 0.83 and r = 0.88, P < 0.05). Iron and Cu were regulated by the white sucker over a wide range of lake pH and metal concentrations in both the water and sediment. In contrast, Zn tissue concentrations were correlated with sediment Zn concentrations, the latter are thought to result from Zn inputs of anthropogenic origin.     

Proteomic Analysis of the Lake Trout (Salvelinus namaycush) Liver Identifies Proteins from Evolutionarily Close and ‐Distant Fish Relatives

Lake trout are used as bioindicators for toxics exposure in the Great Lakes ecosystem. Here the first lake trout (Salvelinus namaycush) liver proteomics study is performed and searched against specific databases: (NCBI and UniProtKB) Salvelinus, Salmonidae, Actinopterygii, and Oncorhynchus mykiss, and the more distant relative, Danio rerio. In the biological replicate 1 (BR1), technical replicate 1 (TR1), (BR1TR1), a large number of lake trout liver proteins are not in the Salvelinus protein database, suggesting that lake trout liver proteins have homology to some proteins from the Salmonidae family and Actinopterygii class, and to Oncorhynchus mykiss and Danio rerio, two more highly studied fish. In the NCBI search, 4194 proteins are identified: 3069 proteins in Actinopterygii, 1617 in Salmonidae, 68 in Salvelinus, 568 in Oncorhynchus mykiss, and 946 in Danio rerio protein databases. Similar results are observed in the UniProtKB searches of BR1RT1, as well as in a technical replicate (BR1TR2), and then in a second biological replicate experiment, with two technical replicates (BR2TR1 and BR2TR2). This study opens the possibility of identifying evolutionary relationships (i.e., adaptive mutations) between various groups (i.e., zebrafish, rainbow trout, Salmonidae, Salvelinus and lake trout) through evolutionary proteomics. Data are available via the PRIDE Q2 (PXD011924).     

Three species of fishes from an eutrophic, seasonally alkaline lake are not more tolerant to acute exposure to high pH in the laboratory

A number of different freshwater fish species (perch Perca fluviatilis , roach Rutilus rutilus and rudd Scardinius erythrophthalamus ) from either eutrophic (Slapton Ley, a seasonally alkaline lake) or non-eutrophic waters were compared with respect to their sodium uptake kinetics and tolerance to acute (1 h) exposure to pH 9·5. Further comparisons were made with rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta . The influence of fish size was also investigated in rainbow trout. Exposure to pH 9·5 was found to disrupt sodium balance and inhibit ammonia excretion in all species and sizes of fishes. The origin of fishes did not have a significant effect on the sodium uptake kinetics or the physiological responses to high pH water. The fishes from the eutrophic lake therefore did not appear to have any increased tolerance to acute exposure to alkaline water. In contrast to previous studies there was no inhibition of Na⁺ uptake during exposure to high pH. Indeed in some groups of fish Na⁺ uptake was actually stimulated, as was Na⁺ efflux. These differences are attributed to experimental water composition and interspecific differences in physiology. It was not always possible to size-match fishes of the different species, so rainbow trout were used to assess the effect of body mass (from 2 to 40 g), on Na⁺ uptake kinetics and Na⁺ or ammonia fluxes during alkaline water exposure in rainbow trout. Size had no significant effect on these measurements within this narrow range, which helps validate the comparison between species in this study.     

Development of radioimmunoassay for thromboxane B2

A simple method for the preparation of rat liver urate oxidase is described. The enzyme was purified from rat liver homogenate by cell fractionation, detergent treatment, alkali treatment, and affinity chromatography on 8-aminoxanthine-bound Sepharose 4B. This enzyme preparation had a specific activity of 9.1 U/mg of protein and was purified about 1000-fold from the liver homogenate. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue, this preparation yielded one protein band at a position corresponding to a molecular weight of 33,000.     

Rainbow trout, Oncorhynchus mykiss, as a model for aromatase inhibition.

The feasibility of utilizing rainbow trout, Oncorhynchus mykiss, as an alternative model for studying the inhibition of aromatase (CYP 19) was investigated. The suppression of estrogen-dependent tumors by aromatase inhibitors has been important in the treatment of breast cancer. Estrogens, estrogen precursors and xenoestrogens have been found to promote liver cancer in the trout model. A steroid, 4-hydroxy-4-androstene-3,17-dione (4-OHA), and non-steroids, aminoglutethimide (AG) and Letrozole (CGS 20267), all of which are known aromatase inhibitors in rats and humans, were examined in vitro for activity in trout ovarian microsomes. Aromatase activity was quantified as the release of 3H2O from the conversion of [3H]-4-androstene-3,17-dione to 17beta-estradiol and estrone. Trout ovarian microsomes exhibited activity between 39-60 fmol mg(-1) min(-1) with a calculated Vmax of 71.1 fmol mg(-1) min(-1) when incubated at 25 degrees C with 200 nM 4-androstene-3,17-dione (K(M) = 435 nM). Significant inhibition by 4-OHA up to 80% was seen at 1.5 microM. At 2000 microM, AG decreased aromatase activity by up to 82%. Letrozole reduced aromatase activity a maximum of 90% in a dose-dependent manner, but the Ki (2.3 microM) was 1000-fold higher than reported in human trials. Indole-3-carbinol and some of its derivatives, two DDE isomers and four flavones (except alpha-naphthoflavone) at 1000 microM did not significantly inhibit aromatase in vitro. Letrozole and clotrimazole, fed to juvenile rainbow trout at doses up to 1000 ppm for 2 weeks, were not effective in suppressing dehydroepiandrosterone (DHEA) induced increases in vitellogenin and 17beta-estradiol levels. These results document that trout aromatase is sensitive to inhibition in vitro by known inhibitors of the mammalian enzyme. The mechanism(s) for lack of inhibition in vivo is currently unknown and must be further investigated in order to develop a trout model for studying the role of aromatase in carcinogenesis.     

Characterization of xenobiotic metabolizing enzymes in sturgeon (Acipenser baeri)

1. Cytochrome P-450, NADPH-cytochrome c reductase, benzo(a)-pyrene hydroxylase (AHH), 7-ethoxycoumarin-O-deethylase (7-ECOD), epoxide hydrolase (EH), UDP-glucuronyltransferase (UDPGT) and glutathione S-transferase (GSHST) activities in sturgeon (Acipenser baeri) have been measured and partially characterized. 2. Cytochrome P-450-dependent monoxygenase (MO), EH, and conjugation reactions were detected in liver and to a lesser extent in kidney and gills. 3. Hepatic enzyme activities in the sturgeon were equally high or higher than in rainbow trout liver, with the exception of UDPGT whose activity was 14% of that in trout liver. 4. The MO and EH activities displayed the expected pH maxima of 7.5, whereas transferases were relatively independent of the pH in the 6.5-7.5 range. 5. The temperature optima for MO and EH were close to those reported in other fish species, whereas for conjugation reactions the temperature optima were 45 and 60 degrees C for GSHST and UDPGT respectively.     

Use of a bacteriophage-derived endo-N-acetylneuraminidase and an equine antipolysialyl antibody to characterize the polysialyl residues in salmonid fish egg polysialoglycoproteins. Substrate and immunospecificity studies

Polysialoglycoproteins (PSGP), a class of glycoproteins containing oligo(poly)sialylglycan chains, are the major glycoprotein components in cortical alveoli of a number of Salmonidae fish eggs. Lake trout, Salvelinus namaycush, egg PSGP (PSGP(Sn)) differs from rainbow trout, Salmo gairdneri, egg PSGP (PSGP(Sg)) in its sialic acid composition; the former contains both N-acetyl- and N-glycolyl-D-neuraminic acid residues, designated Neu5Ac and Neu5Gc, while the latter contains only Neu5Gc residues. Fragmentation analysis of oligo(poly)sialyl chains in lake trout PSGP(Sn) has established that there are two distinct types of oligo(poly)sialyl structures in this PSGP molecule, namely alpha-2,8-linked oligo/poly(Neu5Ac) and alpha-2,8-linked oligo/poly(Neu5Gc). No hybrid structure having both Neu5Ac and Neu5Gc residues in the fragment oligosialic acids was detected. These two distinct PSGP preparations from eggs of lake trout and rainbow trout have been used to compare their immunoreactivity with anti-polysialyl antibodies (H.46) and sensitivity to a bacteriophage-derived (Escherichia coli K1F) endo-N-acetylneuraminidase (Endo-N). H.46 was found to cross-react only with lake trout PSGP(Sn) in immunodiffusion assays but not with rainbow trout PSGP(Sg), indicating that H.46 is a specific probe for alpha-2,8-linked poly(Neu5Ac) but not for poly(Neu5Gc). In contrast, Endo-N was found to catalyze the hydrolysis of both alpha-2,8-linked poly (Neu5Ac) and poly(Neu5Gc), so that this enzyme can be used as a diagnostic reagent for detecting both types of polysialic acids. H.46 was used in indirect immunofluorescence experiments to localize PSGP(Sn) in cortical alveoli isolated from lake trout eggs.     

Proteomic analysis of the lake trout (Salvelinus namaycush) heart and blood: The beginning of a comprehensive lake trout protein database

Lake trout (Salvelinus namaycush) are a top-predator species in the Laurentian Great Lakes that are often used as bioindicators of chemical stressors in the ecosystem. Although many studies are done using these fish to determine concentrations of stressors like legacy persistent, bioaccumulative and toxic chemicals, there are currently no proteomic studies on the biological effects these stressors have on the ecosystem. This lack of proteomic studies on Great Lakes lake trout is because there is currently no complete, comprehensive protein database for this species. Here, we employed proteomics approaches to develop a lake trout protein database that could aid in future research on this fish, in particular exposomics and adductomics. The current study utilized heart tissue and blood from two lake trout. Our previous work using lake trout liver revealed 4194 potential protein hits in the NCBI databases and 3811 potential protein hits in the UniProtKB databases. In the current study, using the NCBI databases we identified 838 proteins for the heart and 580 proteins for the blood tissues in the biological replicate 1 (BR1) and 1180 potential protein hits for the heart and 561 potential protein hits for the blood in BR2. Similar results were obtained using the UniProtKB databases. This study builds on our previous work by continuing to build the first comprehensive lake trout protein database and provides insight into protein homology through evolutionary relationships. This data is available via the PRIDE partner repository with the dataset identifier PXD023970.     

Optimal assay conditions for liver UDP-glucuronosyltransferase from the rainbow trout, Salmo gairdneri

Several kinetic characteristics and assay dependence of UDP-glucuronosyltransferase were studied with microsomal preparations made from liver of rainbow trout. The optimal enzyme assay, performed by incubating less than 5 mg microsomal protein/ml assay buffer for 20 min at 25 degrees C and in pH 7.0, contains 2.5 X 10(-5) M p-nitrophenol (p-NP) and 2.5 X 10(-3) M UDP-glucuronic acid (UDPGA). Apparent Km values revealed that the affinity of trout enzyme for p-NP and UDPGA is, respectively, about 70 and 10 times higher than that of rat enzyme. The optimized method will be used for aquatic bioassays, e.g. when assessing the influencing of toxic effluents from the pulp and paper industry.     

Regiospecificity in the hydroxylation of lauric acid by rainbow trout hepatic cytochrome P450 isozymes

The catalytic activity of two hepatic cytochrome P450 isozymes from untreated rainbow trout towards lauric acid was investigated. In a reconstituted system, cytochrome P450 LMC1 and P450 LMC2 were found to catalyze exclusively the omega- and (omega-1)-hydroxylation of lauric acid, respectively. Microsomal enzyme inhibition studies with polyclonal antibodies raised against the individual P450 isozymes showed that P450 LMC1 and LMC2, respectively, accounted for most if not all the omega- and (omega-1)-lauric acid hydroxylase activity of trout liver microsomes. The polyclonal antibodies were highly specific in that they only inhibited the enzyme activity of the P450 used as the immunogen. These results illustrate that as in mammals, omega- and (omega-1)-hydroxylation of lauric acid by trout liver microsomes can be carried out separately by distinct isozymes of cytochrome P450.     

Long-term trends in limnological characteristics in the Aurora trout lakes, Sudbury, Canada

Paleolimnological techniques were employed to document the limnological histories of the aurora trout lakes, located in the Sudbury region of Ontario. Two of these lakes are of special interest to fisheries managers, as they represent the only known native habitats of a rare strain of brook trout: the aurora trout. These lakes were limed as part of restoration efforts. Stratigraphic changes in diatom and chrysophyte assemblages from dated lake sediment cores indicate that all the lakes have been impacted by anthropogenic acidification, although the timing and the magnitude of acidification were different amongst the lakes. For example, Whirligig Lake was likely the most naturally acidic lake in the past, but it had further acidified since about 1960. This lake was limed in 1989 and then again in 1993. In Whitepine Lake, acidification started 1940; however, in the most recent sediments (1992), some recovery in lakewater acidity has occurred. In Little Whitepine Lake (a reference lake), acidification started earlier (1920) and the lakewater pH continued to decline until about 1990. This lake was limed in 1989. The chrysophyte paleoindicators suggest a recent recovery in this lake. The successful re-introduction of aurora trout in Whirligig and Whitepine lakes is undoubtedly related to the improved water quality through liming but, based on our paleolimnological indicators, the lakes' limnological characteristics (e.g. pH and metal concentrations) are still different from those present before atmospheric deposition of strong acids from the Sudbury smelters.     

Temperature and enzyme activity in poikilotherms. Isocitrate dehydrogenases in rainbow-trout liver

1. The kinetics of the thermally induced enzyme variants of the supernatant NADP-isocitrate dehydrogenase from rainbow-trout liver are investigated. 2. Fish acclimatized to 2 degrees C (cold-adapted enzyme) and 17 degrees C (warm-adapted enzyme) show different relative distributions of the three NADP-isocitrate dehydrogenase isoenzymes; this has been demonstrated with electrophoresis and electrofocusing techniques. 3. Plots of K(m) versus temperature for the cold-adapted and warm-adapted enzyme variants are complex in nature with apparent maximal enzyme-substrate affinity corresponding to the temperature at which the trout is acclimatized. Both substrates, dl-isocitrate and NADP(+), give similar curves although the magnitude of the K(m) change with temperature is much decreased in the case of NADP(+). 4. E(a) values of approx. 18kcal/mol were determined for both the cold-adapted and warm-adapted enzyme variants. 5. In an attempt to determine how velocities can be increased at low temperatures, cation, pH requirements, metabolite and enzyme concentrations were examined. 6. NAD-isocitrate dehydrogenase could not be detected in trout tissues.     

The stimulation of liver phosphorylase b by AMP, fluoride and sulfate. A technical note on the specific determination of the a and b forms of liver glycogen phosphorylase.

1. The activity of liver phosphorylase b from several mammalian species has been studied. The enzyme from rat or mouse has a higher activity than the rabbit enzyme, which is itself more active than pig liver phosphorylase b. 2 The activity of liver phosphorylase b is influenced by anions and by AMP, and these effects are influenced by pH. Fluoride, which is currently added to the assay mixture of phosphorylase a in crude preparations, is about as active as sulfate as a stimulator of phosphorylase b. 3. When assayed at pH 6.1 and in the presence of 0.15 M NaF, the activity of rat liver phosphorylase b reaches 25% of that of the a enzyme; if 1 mM AMP is also present, this value rises to 50%. 4. Methods are described that allow the determination of liver phosphorylase a without interference of b, and the determination of total phosphorylase (a+b) in rat liver.     

Isolation and characterization of porcine parathyroid cathepsin B.

Cathepsin B was isolated from porcine parathyroid tissue and from liver by a procedure involving acetone precipitation, gel filtration, and carboxymethylcellulose chromatography. The final preparations of each migrated as single bands upon sodium dodecyl sulfate polyacrylamide gels but exhibited several minor active variants upon isoelectric focusing. The parathyroid and liver enzymes were similar to each other and also resembled cathepsin B from other sources. The molecular weights for the porcine enzymes were estimated as 25,000, and the isoelectric point was at pH 4.8. The parathyroid enzyme cleaved benzyloxycarbonyl-Val-Lys-Lys-Arg-(4-methoxy)-2-naphthylamide at pH 5.8 and 37 degrees C with a Km of 0.14 mM and a kcat of 68 s-1. The pH optimum for this reaction was pH 6 to 7. The enzyme was unstable above pH 7.5 and below pH 4.5. It was strongly inhibited by HgCl2, ZnSO4, iodoacetate, iodoacetamide, and N-ethylmaleimide which indicated that it is a thiol protease, and by leupeptin, a strong inhibitor of cathepsin B from other sources. Antibodies to the parathyroid enzyme were elicited in rabbits. The antisera formed single precipitin bands upon double diffusion in agar gels against both the parathyroid and liver enzymes. Precipitin bands were formed at both pH 6 and pH 8.5 which indicated that the antisera recognized both native and denatured forms of the enzymes.     

The structural basis of anomalous kinetics of rabbit liver aryl sulfatase A

Rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) is inactivated during the hydrolysis of nitrocatechol sulfate and the rate of formation of turnover-modified aryl sulfatase A depends on the initial velocity of the enzymatic reaction. Organic solvents such as ethanol and dioxane favor the anomalous kinetic behavior. The turnover-modified enzyme can apparently be reactivated by arsenate, phosphate, pyrophosphate, and sulfate in the presence of nitrocatechol sulfate. The apparent dissociation constants of these ions in the reactivation of the enzyme are similar to their K_i values. Sulfite, which is a competitive inhibitor, does not reactivate the turnover-modified enzyme. Thus, all known activators are competitive inhibitors but not all competitive inhibitors are effective as activators. Inactivation of aryl sulfatase A during hydrolysis of ³⁵S-labeled substrate at pH values near the pH optimum (pH 5–6) is accompanied by the incorporation of radioactivity into the protein molecule and the turnover-modified enzyme is thereby covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of sulfur per mole of enzyme monomer, or 1 g atom of sulfur per equivalent peptide chain. It is also shown that isolated turnover-modified rabbit liver aryl sulfatase A has lost approximately 76% of its secondary structure as compared to the native enzyme. The specific activity of the inactive enzyme is also decreased by 82%. Turnover-modified rabbit liver aryl sulfatase A is partially reactivated by sulfate ions in the presence of nitrocatechol sulfate. However, circular dichroism measurements and fluorescence spectra of the isolated “reactivated” turnover-modified enzyme indicate only a further loss of secondary structure. The specific activity of this “reactivated” enzyme is in fact decreased. The loss in secondary structure and the enzyme activity of the “reactivated” aryl sulfatase A is prevented in the presence of sulfate ions. Turnover-modified rabbit liver aryl sulfatase A behaves as a very fragile molecule.