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Identification and initial characterization of serum growth hormone binding protein in the turtle Chrysemys dorbigni
A I Sotelo1, W A Partata, M Marques
1Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Insights
Researchers identified growth hormone-binding proteins (GHBPs) in turtle serum, characterizing their high-affinity binding properties and seasonal activity variations. This study expands the known distribution of GHBPs in vertebrates.
Area of Science:
- Comparative Endocrinology
- Biochemistry
- Zoology
Background:
- Growth hormone-binding proteins (GHBPs) are crucial for regulating growth hormone (GH) bioavailability.
- GHBPs have been identified across mammalian and avian species, but their presence in reptiles remained unconfirmed.
Purpose of the Study:
- To investigate the presence and characteristics of GHBPs in reptilian species, specifically turtles.
- To determine the binding affinity, capacity, molecular weight, and specificity of turtle GHBP.
- To explore potential seasonal variations in turtle GHBP activity.
Main Methods:
- Serum samples from turtles were analyzed using chromatographic techniques and dextran-charcoal separation.
- High-performance gel filtration and affinity chromatography were employed for partial purification of high-affinity GHBP.
- Binding studies were conducted to determine dissociation constant (Kd), binding capacity (Bmax), and hormone specificity.
- Preliminary molecular weight (MW) estimation was performed.
Main Results:
- Two distinct GHBPs were identified in turtle serum: one high molecular weight (MW) with low affinity, and another lower MW with high affinity.
- The high-affinity GHBP was purified over 11,000-fold, exhibiting a Kd of 2.6 +/- 0.7 nM and Bmax of 120 +/- 43 fmoles/mg protein.
- Preliminary MW estimation for the high-affinity GHBP was 50-60 kDa, with somatogenic specificity.
- Significant seasonal variation in GHBP activity was observed, peaking in November.
Conclusions:
- This study confirms the presence of GHBPs in a reptilian species (turtle) for the first time.
- Turtle GHBP shares characteristics with mammalian GHBPs but exhibits distinct binding affinities and seasonal variations.
- The findings contribute to understanding the evolution and function of GHBP across vertebrate classes.
Abstract:
Proteins that bind growth hormone (GHBP) have been identified in the blood of many mammalian and avian species, but not in reptilian species. We carried out binding studies with the serum of turtles using chromatographic techniques as well as the dextran-charcoal separation method. As in other species, we found at least two different GHBPs: one with high MW and low affinity and the other with lower MW and higher affinity. The high affinity GHBP was partially purified using gel filtration and affinity chromatography, reaching a degree of purification of 11,000 times (0.17 nmol/g of serum protein in the serum vs 1900 nmol/g protein in the purified material). When the high affinity GHBP was characterized, it was found to have a dissociation constant (Kd: 2.6 +/- 0.7 nM) similar to those described for mouse or rat, but lower than those for chicken, rabbit or man. The binding capacity (Bmax) was 120 +/- 43 fmoles/mg of protein, which can be also expressed as 1.08 +/- 0.38 pmol/ml of serum. A preliminary MW estimation of 50-60 kDa was obtained for turtle higher affinity GHBP. The specificity of this high affinity GHBP is somatogenic, since bovine GH competes as well as human GH for 125I-hGH bound to binding protein, while ovine PRL competes only partially and with low affinity. Unrelated hormones, as insulin and glucagon, can not displace the 125I-hGH bound to turtle GHBP. A very important seasonal variation in turtle GHBP activity was observed: maximum binding was found in November (springtime), followed by a continuous decline over March and May.