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1.
The aim of this work was to verify if, compared to mammals, the lower molecular weight of GH-R previously reported in salmonid is real or due to the experimental process. For this purpose, we compared the apparent molecular weight of GH-R, obtained by SDS-PAGE after cross-linking with 50 I-rtGH, obtained from rainbow trout crude liver membrane preparation, incubated in different buffers with those obtained after purification with affinity chromatography.Using crude liver membrane preparation, two specific bands of 50 I-rtGH-protein complex were observed: the major one corresponds to a MW of 70 kDa and the minor one to 45 kDa. However, the pattern of electrophoresis varied according to the different incubation buffers tested. Digestion of the cross-linked complex with -galactosidase and phospholipase did not significantly modify the position of bands, whilst N-glycosidase F induced a large smear including 4 more pronounced bands (50, 65, 97 and > 130 kDa), the heavier band corresponding to the most intensive signal.GH receptors were purified using solubilisation and affinity chromatography. The yield of the liver GH-R from crude liver membrane preparation by the solubilization technique was optimized (48%) using Triton 1% for 1 h (12°C ). Specific binding sites in the solubilized membrane proteins were saturable when incubated with increasing 50 I-rtGH concentrations, and revealed a high affinity constant (Ka=0.7×109 M–1). After affinity chromatography, specific binding activity was increased 64,000 fold. However, the purity of the preparation was partial and the purification yield was very low (about 0.3%). This enriched fraction, analysed by SDS-PAGE after cross-linking, showed a very intense band (about 63 kDa) which disappeared with an excess of cold rtGH.These results suggest that the lower molecular weight observed in salmonid (41 kDa), compared to mamals, is not due to the experimental process. The significance of GH-R size difference between salmonids and mammals is discussed.  相似文献   

2.
大鳞大马哈鱼生长激素抗体的制备与鉴定   总被引：1，自引：0，他引：1  
本文用大鳞大马哈鱼生长激素（ｓＧＨ）免疫日本大耳白兔制备了特异抗血清。应用常规酶联免疫吸附测定法（ＥＬＩＳＡ）对抗血清效价和特异性进行了测定，结果表明ｓＧＨ抗血清只与ｓＧＨ特异反应，与草鱼ＧＨ、草鱼催乳素（ＰＲＬ）、牛ＧＨ及大群大马哈鱼促性腺激素（ｓＧｔＨ）等基本上没有交叉反应，该抗血清的效价约为１：７００００左右。应用ｓＧＨ抗血清，初步建立了ｓＧＨＥＬＩＳＡ检测方法。  相似文献   

3.
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4.
Most previous studies of the temperature-induced restructuring of phospholipid molecular species composition have examined steps in the biosynthesis of phospholipids to explain the accumulation of unsaturated fatty acids in membranes of cold-acclimated poikilotherms. In contrast, the present study explores the role of phospholipases in this restructuring process by determining the rates of degradation of specific molecular species of phosphatidylcholine, using enzymes (microsomes) freshly isolated from the liver of rainbow trout. (Oncorhynchus mykiss) acclimated to either 5° or 20°C. The substrate preparation employed to assay phospholipase activity possessed a range of molecular species, all radiolabeled with 1-14C-palmitic acid at thesn-1 position, similar to that present in native trout liver microsomes. After defined periods of incubation (120 and 240 min at 5°C; 60 and 120 min at 20°C), phospholipids were extracted from the reaction mixture and the distribution of radioactivity among the molecular species of phosphatidylcholine was determined by HPLC/liquid scintillation counting. In general, molecular species catabolism was not significantly influenced by either assay or acclimation temperature. Only in 20°C-acclimated fish did a reduction in assay temperature (from to 20 to 5°C) result in significantly increased proportions of radioactivity being recovered in one polyunsaturated fatty acid-containing species (16:0/22:6-PC). It is concluded: 1) that phospholipase specificity, assayed under conditions approximating thosein situ, is not significantly influenced by temperature; and 2), that the increased proportions of unsaturated fatty acid-containing molecular species of phosphatidylcholine observed at low temperatures must reflect the specificity of biosynthetic rather than degradative processes.  相似文献   

5.
In fish, regulation of the growth hormone (GH) receptor has been mainly analyzed by binding studies, with some discrepancies in the results. The present work aims at determining whether circulating GH levels influence the measurement of hepatic GH-binding capacities. To do this, the effectiveness of the dissociation of the GH/GH receptor (GHR) complex by an MgCl2 treatment was assessed in rainbow trout, and data on GH-binding capacities under various physiological conditions were studied using different means of expression. Our results reveal that MgCl2 treatment dissociated the liver GH/GHR complex formed under in vitro conditions (85 ± 23 vs. 361 ± 16 fmol/g of liver; p<0.001) but not in vivo, showing such treatment in trout is not applicable. A comparison of fasted (3 weeks) and fed fish revealed that GH-binding capacities, expressed as femtomoles per milligram protein or femtomoles per gram of liver, were similar in both fed and fasted fish. However, when changes in liver and body size were taken into account, the total GH-binding capacities were lower in the fasted fish (0.026 ± 0.006 vs. 0.062 ± 0.009 fmol/cm3 liver; p<0.05). One day after hypophysectomy or GH injection, changes in the plasma GH levels increased or decreased GH-binding capacities, respectively. Five days later, GH-binding capacities increased in GH-injected fish (527 ± 38 vs. 399 ± 38 fmol/g liver; p<0.01). Our interpretation is that acute treatment modified GH-binding capacities through receptor occupancy and that GH stimulated the synthesis of its own receptor. On the other hand, long-term treatment through successive injections of GH lowered the total binding capacities (approx. 40%), which could result from receptor occupancy. We conclude that circulating GH levels strongly influence the measurement of GH-binding capacities in the liver, thereby limiting interpretation of the binding data and preventing accurate conclusions to be drawn on GHR regulation.  相似文献   

6.
Liver is the main catabolic tissue for low density lipoprotein in rainbow trout (Gjøen and Berg 1992). We have investigated the interaction of LDL with isolated trout liver cells and liver membranes. 125I-TC labelled trout LDL bound to isolated trout liver cells in a time dependent and saturable manner with an apparant Kd of 20.1 g/ml, suggesting the existence of a specific binding site on the surface of these cells. The binding was Ca2+ dependent assessed by the 50% reduction obtained by 5 mM EDTA. Saturable binding to isolated trout liver membranes could also be demonstrated, but with lower affinity as compared to intact cells. Degradation of 125I-TC-LDL in hepatocytes was also saturable as degradation could be inhibited about 60% by a 100 fold surplus of unlabelled LDL. The rate of degradation increased with temperature up to 20°C. Both cell association (binding + uptake) and degradation were reduced down to 20% of control in the presence of microtubular and lysosomal inhibitors. Hepatic catabolism of trout LDL therefore seems to depend on receptormediated endocytosis, followed by lysosomal degradation.Abbreviations TC tyramine cellobiose - LDL low density lipoproteins - MeLDL methylated low density lipoproteins - VLDL very low density lipoproteins - HDL high density lipoproteins - VTG vitellogenin - EDTA ethylenediamine tetraacetic acid - PBS phospate buffered saline - SDS-PAGE sodium dodecyl sulphatepolyacrylamide gel electrophoresis - DMPC L--phosphatidylcholine-dimyristoyl  相似文献   

7.
In saffron cod, Eleginus gracilis, the antifreeze glycoprotein (AFGP) is present during the winter. The effects of salmon prolactin (sPRL) and growth hormone (sGH) on plasma osmolality, sodium concentration and protein content of the saffron cod were investigated. In the winter, plasma osmolality and protein content decreased following the intraperitoneal injection of sPRL (20 µg kg-1 day-1 × 3) but injection of the same dose of sGH had no effect on either plasma osmolality or protein content. In similar experiments performed during the summer, sPRL and sGH had no effect on these parameters. The results suggest that PRL may act on the kidney, possibly clearing antifreeze glycoprotein (AFGP) by increasing glomerular filtration. The possible role of PRL and GH in the disappearance of AFGP during the summer are discussed.  相似文献   

8.
Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring14C-oleic acid release from14C-triolein.14C-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15°C. Most of the lipase activity was recovered in the cytosolic fraction. A 27,000-fold purification was achieved after Sepharose (Bio-gel A 0.5 M, 200–400 mesh) chromatography of a resuspended 20% ammonium sulfate fraction. The molecular weight of the trout hepatic lipase as determined by size-exclusion chromatography and by SDS-polyacrylamide gel electrophoresis was 40–43 kD. Lipase-mediated hydrolysis of TG resulted in the production of diacylglycerols, monoacylglycerols, and fatty acids. Kinetic analysis indicated that Vmax=0.016 nmol/h/mg protein and that Km=0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg2+. These results suggest that the liver of trout possesses a neutral TG lipase that is responsible for mobilizing stored TG and is catalytically activated by phosphorylation.A part of this work was presented at the Annual Meeting of the American Society of Zoologists, December 26–30, 1990, San Antonio, TX.  相似文献   

9.
Two isoforms of the full-length cDNA of the growth hormone receptor (GHR) of the Atlantic salmon (Salmo salar; ss) were cloned by a PCR approach using RACE. Respectively, the cDNA sequences of ssGHR isoforms 1 and 2 are 2654 and 2608 nucleotides long, with 1782 and 1773 nucleotide ORFs. The resulting coded proteins are 594 and 590 aa long, with 19 and 20 aa signal peptides. The two isoforms share 86% protein and 87% cDNA sequence similarity. Isoform 1 is most similar to other salmonid GHR isoforms 1 while isoform 2 is most similar to salmonid GHR isoforms 2 (93–95%). Similarity with other teleost species was lower (37–44%). The bioactivity of the cloned ssGHR was tested by transfecting the ssGHR isoform 1 cDNA into CHO-K1 hamster cells, incubating with recombinant salmon GH (sGH) or native ovine prolactin (oPRL), and measuring cell proliferation by the MTT assay. The ssGHR-transfected cells significantly increased proliferation when stimulated by sGH at all concentrations. oPRL stimulated ssGHR-transfected cells at higher concentrations due to receptor cross reaction. ssGHR isoforms 1 and 2 contain a single transmembrane domain and the typical conserved motifs found in other teleost GHRs, including four paired cysteine residues and five potential N-glycosylation sites in the extracellular domain, Box I and Box II, as well as seven potential tyrosine phosphorylation sites in the intracellular domain. However, in salmonids, these motifs differ from those of other teleosts, and could be responsible for differentiated hormone binding, signal transduction and response.  相似文献   

10.
This paper reports on the effect of administration of mammalian growth hormone (GH) on muscle protein synthesis as measured in white muscle using the phenylalanine flooding technique. The effect of exogenous GH was compared with that of insulin and prolactin, and with endogenous GH.The rate of protein synthesis in white muscle of rainbow trout 6 h after the injection of bovine GH or bovine insulin was twice (2.6 and 2.9% d–1) that of the control saline-injected fish (1.2% d–1). A metabolic effect of GH, as observed with insulin, is suspected.The rates of change in body weight and body length and the fractional rate of protein synthesis in muscle of rainbow trout were enhanced by mammalian GH administration. The effect of GH on muscle RNA/protein ratios was not significant. An opposite effect of antibodies against salmon GH (Lebailet al. 1989) on growth rate and muscle protein synthesis rate was found in rainbow trout. It is suggested that the effects of exogenous and endogenous GH on capacity and efficiency of muscle protein synthesis were similar.The long-term effects of mammalian GH on presmolt Atlantic salmon was also tested. The same trends were found with ovine prolactin supplementation in Atlantic salmon but not as high as those observed with ovine GH.  相似文献   

11.
Glutathione and its Related Enzymes in the Nile Fish   总被引：2，自引：0，他引：2  
Glutathione (GSH) and related enzymes, glutathione transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) form an important phase 2 biotransformation enzymes system. The objective of this study was to compare this enzymes system in three fish species from the river Nile, Oreochromis niloticus, Claris lazera and Cyprinus carpio in order to establish the main differences and to purify and characterize GST from the liver of O. niloticus.The level of GSH and the activity of GST, GPx and GR in the liver, kidney and gills of the three fish species were examined. A simple reproducible procedure for the purification of GST from the liver of O. niloticus to homogeneity, which includes chromatography on DEAE- cellulose followed by affinity chromatography on GSH-sepharose was established. The molecular mass was found to be 25,460 Da by SDS-PAGE. The Michaelis-Meneten constants (Km) of the enzyme for GSH and CDNB were 0.35 mM and 0.42 mM, respectively. The affinity purified enzyme exhibited maximum pH at pH 8.0 and increasing pH above 8.0 did not affect the observed maximum. The purified enzyme acts readily on CDNB, less readily on some standard transferase substrates (1,2-dichloro-4-nitrobenzene and p-nitrophenethyl bromide) and not at all on others (bromosulphophthalein and p-nitrobenzyl chloride). Bromosulfophthalein, cibacron blue and hematin inhibited CDNB-conjugating activity of the purified enzyme with IC50 0.079, 3.98 and 0.126 μM, respectively.  相似文献   

12.
The association between the presence of EGF binding and its associated tyrosine kinase activity was investigated in the hepatic membranes of rainbow trout (Oncorhynchus mykiss). Time course experiments indicted that the binding of EGF to hepatic membranes was temperature dependent and resulted in increased phosphorylation of membrane components. The phosphorylation reaction was dependent on the presence of divalent ions (Mg2+ and Mn2+) but not monovalent ions (Na+ and K+) or calcium. Western blot analysis of phosphorylated trout membranes indicated protein bands at 150 and 180 kDa which is similar to that observed in mammals. Use of specific substrates and protein kinase inhibitors also indicated that the increase in EGF stimulated phosphorylation was related to an increase in tyrosine kinase activity. These data support the hypothesis that trout have an EGF-like receptor that has binding and biological activities similar to that observed in mammalian species.  相似文献   

13.
Fish growth hormones (GHs) play an important role in regulating growth, metabolism, reproduction, osmoregulation, and immunity and have thus garnered attention for their application in aquaculture. Zebrafish GH (zGH) cDNA or rainbow trout GH (rtGH) cDNA was cloned into the pMon3401 vector, expressed in MON105-competent Escherichia coli and purified to homogeneity. Their biological activity was evidenced by their ability to interact with ovine GH receptor extracellular domain and stimulate GH receptor-mediated proliferation in FDC-P1-3B9 cells stably transfected with rabbit GH receptor. The relative affinity of zGH and rtGH, estimated by IC50, was about 38-fold and 512-fold lower, respectively, than ovine GH. This is likely the reason for the low biological activity in cells with rabbit GH receptor, ~?36-fold lower for zGH and ~?107-fold lower for rtGH than for human GH. This was not due to improper refolding, as evidenced by circular dichroism analysis. Predicting the activity of fish GHs is problematic as there is no one single optimal in vitro bioassay; heterologous assays may be ambiguous, and only homologous assays are suitable for measuring activity.  相似文献   

14.
A method for purification of rainbow trout, Oncorhynchus mykiss, serum immunoglobulin (Ig) that relies on specific retention of rainbow trout Ig in the presence of other serum proteins, by an ion-exchange chromatography matrix (ABx resin) previously developed for the isolation of mammalian Ig is described. This protocol allows for rapid and substantial purification of total Ig including antigen-specific antibodies from the serum of rainbow trout. The procedure does not utilize antibody-derivatized matrices for immunoabsorption so it is suited to situations where fish Ig-specific antisera or monoclonal antibody is unavailable. Furthermore, total Ig can be isolated from pre-immune sera since no antigen-derivatized affinity matrices are used, eliminating the requirement for lengthy immunization regimens. With minor modification, ABx-chromatography can be adapted for the isolation of Ig from other species of fish.  相似文献   

15.
ABSTRACT

The objectives of this study were to characterize physicochemical properties of two collagens, tilapia skin (TS) and hybrid catfish skin (HS). Angiotensin-converting enzyme (ACE) inhibitory peptides from extracted TS and HS collagen using pepsin were also determined. HS collagen had a higher amount of imino acid than TS collagen, while TS contained higher amounts of tyrosine (Tyr) and lysine (Lys). Fourier-transform infrared spectra of both collagens showed predominant helix structure. The HS collagen hydrolysate prepared by pepsin was fractionated using sequential ultrafiltration membranes, and the fraction with molecular weight (MW) <5 kDa showed the highest ACE inhibitory activity (p < .05). After cation exchange and two steps size exclusion chromatography, peptides showed ACE inhibitory activity of 72.06%. This study revealed that ACE inhibitory peptides derived from HS collagen could be developed as a functional food with potential antihypertensive properties.  相似文献   

16.
Although fish feed supplemented with Chinese parsley (CP), Coriandrum sativum, does not affect the health of rainbow trout Oncorhynchus mykiss, it has the remarkable effect of reducing the uptake of Cd (12–17% compared with controls without CP). Here, we measured the change in the amount of metallothionein (MT) in the liver and kidney using gel permeation chromatography-high-performance liquid chromatography (TSK GEL SW 3000 column). The increase in MT content over time in the Cd-fed groups corresponded to the change in Cd accumulation. Gel permeation was performed with soluble fractions from the fish liver and kidney, and changes in the Cd, Cu, and Zn concentrations over time for each fraction, and the relationship between the form of Cd that accumulated in tissues and its toxicity were measured. A nontoxic MT fraction of 7,000 Da and a high-molecular-weight fraction of approximately 60,000 Da were isolated from the soluble fractions. The induction of nontoxic MT–Cd was higher in the CP-supplemented groups than in the control groups. The synergetic actions of several compounds in CP may contribute to bind MT, thereby preventing accumulation of harmful heavy metals and essential metals in fish.  相似文献   

17.
Recently, a rapid increase in the resistance of pathogenic bacteria to antibiotics and chemotherapeutics admitted for use in aquaculture has been observed. This happens especially often in intensive breeding. The use of drugs in closed circuits is problematic because it can damage biological filters. Therefore, in recent years, there has been a growing interest in natural methods of combating pathogens. These include bacteriophages. The aim of the study was to determine the safety of the new BAFADOR® bacteriophage‐based preparation, its effect on selected immunological parameters and the effectiveness of prophylactic and therapeutic use after experimental infections with pathogenic bacteria Aeromonas hydrophila and Pseudomonas fluorescens. The use of BAFADOR® increased the activity of lysozyme, total protein level and immunoglobulin level. The level of ceruloplasmin in the rainbow trout serum remained unchanged regardless of the route of administration of the preparation. Potential killing activity and metabolic activity of spleen phagocytes and proliferation of pronephros lymphocytes were higher compared to the control group. Both therapeutic and prophylactic application of the preparation after mixed experimental infection of A. hydrophila and P. fluorescens limited the mortality of rainbow trout.  相似文献   

18.
The plasma inhibitor of carbonic anhydrase (CA) from rainbow trout was purified using ion exchange chromatography. The inhibitor has a high isoelectric point value (pI>10). SDS-PAGE electrophoresis and gel filtration demonstrated that the inhibitor is a low molecular weight compound of about 6,000 daltons. The plasma inhibitor was more effective against gill CA than against blood CA in vitro, probably reflecting the presence of various CA-isoenzymes in red blood cells and gill tissue. The apparent Root effect, i.e., the impairment of the oxygen binding capacity of hemoglobin in red blood associated with increased blood PCO 2was counteracted by the plasma inhibitor, probably by acting on membrane-bound and/or cytosolic blood CA. This interaction may be of importance in adaptive mechanisms, e.g., during the acidemic phase, when the fish is being acclimated to hypercapnic conditions.  相似文献   

19.
We have studied the seasonal relationship between growth and circulating growth hormone (GH), hepatic GH-binding and plasma insulin-like growth factor-I immunoreactivity in gilthead sea bream,Sparus aurata. The seasonal increase in plasma GH levels preceded by several weeks the summer increase in growth rates. In contrast, a marked increase in hepatic GH-binding with a high degree of endogenous GH occupancy was found during the period of maximum growth which suggests an enhanced sensitivity of liver to GH action. Thus, circulating levels of immunoreactive IGF-I, probably derived from the liver in response to GH action, were positively correlated with growth throughout the experimental period although a consistent relationship between growth and circulating GH was not found. In spite of this, we consider that, in gilthead sea bream, as in several other teleosts, the availability of endogenous GH can limit growth. Thus, under environmental conditions of suboptimal growth, a single intraperitoneal injection of recombinant rainbow trout GH (rtGH) induced over the dose range tested (0.75, 1.5, 3 μg g BW−1) an increase in plasma IGF-I-like immunoreactivity comparable to that seen during the period of maximum growth.  相似文献   

20.
Gastric uptake of recombinant growth hormone in rainbow trout   总被引：1，自引：0，他引：1  
The aim of the present study was to investigate gastrointestinal uptake of recombinant growth hormone in rainbow trout. Recombinant human GH was used in this study to avoid cross reactivity with the endogenous trout GH molecule. Using both in vitro and in vivo approach, the results demonstrate that stomach is the main site of uptake for orally administered intact recombinant human growth hormone (hGH) in rainbow trout. The presence of hGH was verified by western blot analysis in the stomach tissue as well as in the serum after 30 minutes of intubation. No traces of the hGH could be detected after 90 minutes of administration, indicating rapid clearance of the hormone in rainbow trout. Furthermore, no immunoreactive hGH was detected in other tissues including mid-gut, muscle and liver at the time points tested. The findings provide novel information on the gastric uptake of hGH in rainbow trout, and would be of relevance to the application of GH in aquaculture.  相似文献   

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