Characterization of growth hormone binding sites in the goldfish,Carassius auratus: effects of hypophysectomy and hormone injection

Zhang, Yushi; Marchant, Tracy A.

doi:10.1007/bf01875595

Cited by 9 publications

(14 citation statements)

References 37 publications

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“…This change was necessary as the optimal binding of 125 I-rcGH to all serum-binding proteins, defined as the highest specific binding with a non-specific binding of less than 5%, was found to occur at pH 6·5 (data not shown). This is similar to our previous finding for the binding of 125 I-rcGH to goldfish liver membranes (Zhang & Marchant 1996). Maximal binding of 125 I-bGH to rabbit and rat serum was found to occur at pH 7·2, but was only slightly reduced (<1%) at pH 6·5, with no alteration in the shape of the 125 I-bGH displacement curve (data not shown).…”

Section: Iodination and Binding Studiessupporting

confidence: 91%

“…Goldfish of the common or comet varieties were maintained as previously described (Zhang & Marchant 1996). The goldfish were anesthetized by immersion in 0·005% (w/v) tricaine methanesulfonate (MS222).…”

Section: Experimental Animalsmentioning

confidence: 99%

“…Iodination of the GHs was performed as previously described, using the lactoperoxidase method (Zhang & Marchant 1996). The specific activity of the labeled hormones was routinely more than 100 µCi µg -1 when determined using self displacement (Zhang & Marchant 1996) in liver membrane receptor-binding assay.…”

Section: Iodination and Binding Studiesmentioning

confidence: 99%

“…The specific activity of the labeled hormones was routinely more than 100 µCi µg -1 when determined using self displacement (Zhang & Marchant 1996) in liver membrane receptor-binding assay.…”

Section: Iodination and Binding Studiesmentioning

confidence: 99%

“…The fraction of 125 I-rcGH, 125 I-bGH and 125 I-rsbGH that would bind to an infinite concentration of serum protein was calculated to be approximately 80% of the total radioactivity added. Therefore, total binding was corrected prior to LIGAND analysis as described previously (Zhang & Marchant 1996). Typical elution profiles for the total and non-specific binding of 125 I-rcGH to goldfish serum are shown in Fig.…”

Section: Gh-binding Studiesmentioning

confidence: 99%

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Identification of serum GH-binding proteins in the goldfish (Carassius auratus) and comparison with mammalian GH-binding proteins

Zhang

Marchant²

1999

Journal of Endocrinology

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The present study constitutes the characterization of a specific, high-affinity GH-binding protein (GHBP) in the serum of a teleost, the goldfish (Carassius auratus). GH-binding assay and ligand blotting techniques were employed to identify GHBPs in goldfish serum and hepatocyte culture medium. The binding characteristics and apparent molecular weights (M r ) of goldfish GHBPs were also compared with those of rabbit and rat. LIGAND analysis identified a single class of high-affinity and lowcapacity binding sites for iodinated recombinant carp GH (rcGH) in the goldfish serum, with an association constant (K a ) of 20·1 10 9 M 1 and a maximum binding capacity (B max ) of 161 fmol ml 1 serum. A single class of binding sites for iodinated recombinant sea bream GH and bovine GH (bGH) was also found in goldfish serum, but with a much lower affinity than that of rcGH. The binding affinity for iodinated bGH in rabbit and rat sera was found to be similar to that reported previously. Ligand blotting revealed multiple forms of GHBPs in sera of goldfish, rabbit and rat with M r ranging from 70 kDa to 400 kDa and 27 kDa to 240 kDa under non-reducing and reducing conditions respectively. A prominent band with M r of 66 kDa and a minor band with M r of 27 kDa were observed to occur in sera from all three species under reducing conditions. Iodoacetamide promoted the shedding of three GHBPs with M r of 25, 40 and 45 kDa from the cultured goldfish hepatocytes. The appearance of all bands was completely inhibited by the presence of excess unlabeled rcGH. Our results provide clear evidence that a GHBP exists in the goldfish and indicate that more information on teleost GHBPs is needed if the physiology of growth in teleosts is to be fully understood.

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Section: Iodination and Binding Studiessupporting

confidence: 91%

Section: Experimental Animalsmentioning

confidence: 99%

Section: Iodination and Binding Studiesmentioning

confidence: 99%

Section: Iodination and Binding Studiesmentioning

confidence: 99%

Section: Gh-binding Studiesmentioning

confidence: 99%

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Identification of serum GH-binding proteins in the goldfish (Carassius auratus) and comparison with mammalian GH-binding proteins

Zhang

Marchant²

1999

Journal of Endocrinology

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Regulation of Food Intake by Neuropeptides and Hormones

Pedro¹,

Björnsson²

2001

Food Intake in Fish

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Influence of circulating GH levels on GH-binding capacity measurements in the hepatic membrane of rainbow trout (Oncorhynchus mykiss): importance of normalization of results

Yao

Gabillard²,

Bail³

2006

Fish Physiol Biochem

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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 MgCl 2 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 MgCl 2 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/ cm 3 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 GHbinding 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.

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Characterization of growth hormone binding sites in the goldfish,Carassius auratus: effects of hypophysectomy and hormone injection

Cited by 9 publications

References 37 publications

Identification of serum GH-binding proteins in the goldfish (Carassius auratus) and comparison with mammalian GH-binding proteins

Identification of serum GH-binding proteins in the goldfish (Carassius auratus) and comparison with mammalian GH-binding proteins

Regulation of Food Intake by Neuropeptides and Hormones

Influence of circulating GH levels on GH-binding capacity measurements in the hepatic membrane of rainbow trout (Oncorhynchus mykiss): importance of normalization of results

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