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Covalent cross-linking of the bovine somatotropin dimer. Effects on growth-promoting, receptor-binding and immunological activities and preliminary characterization of the self-association.

ABSTRACT: Bovine somatotropin, at pH 8.5 in 0.02 M-Bicine [NN-bis-(2-hydroxyethyl)glycine]/0.09M-NaCl, showed by frontal analysis the characteristics of a rapid monomer-dimer equilibrium whose dissociation constant was estimated to be 6.6 X 10(-6)M. Reaction of the hormone with dimethyl suberimidate lead to covalent cross-linking of the dimeric species. Under the conditions chosen (0.4 mg of bifunctional imidate and 1 mg of protein/ml at room temperature for 1 h) the cross-linked dimers accounted for 26% of the total protein, and these were isolated by molecular sieving in 0.29M-NH3/0.12M-NaCl. Covalent stabilization greatly diminished the growth-promoting activity and the ability to interact with somatogenic sites in both rat liver in vivo and rabbit liver microsomal fractions. Evidence indicating a non-critical role for amino groups involved in the covalent cross-linking was provided by a nearly equivalent derivative obtained after reaction with 3,3'-dithiobispropionimidate, which had substantial hormonal activity upon cleavage of the disulphide links. Conversely, immunological reactivity as demonstrated by radioimmunoassay was not affected by cross-linking. Details of the least-squares procedure employed to evaluate the self-association equilibrium constant has been deposited as Supplement SUP 50115 (7 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem.J. (1981) 193,5.

SUBMITTER: Fernandez HN

PROVIDER: S-EPMC1154061 | biostudies-other | 1983 Jan

REPOSITORIES: biostudies-other

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Project description:The physiological mechanisms by which growth hormone (somatotropin) exerts its several metabolic activities remain poorly understood. In particular, there is disagreement as to whether the diabetogenic and lipolytic activities of the hormone are intrinsic properties of the molecule or are the result of contamination with other pituitary components. The availability of recombinant-DNA-derived bovine growth hormone (rebGH) presented an opportunity to compare the biological activities of rebGH and pituitary bGH in the absence of pituitary contaminants. Pituitary bGH and rebGH were immunologically identical in the radioimmunoassay for bGH, and good agreement was obtained for the potency of the latter measured by radioimmunoassay (1.6 units/mg) and the dwarf-mouse bioassay (1.4 units/mg). The lipolytic activity in vitro was examined by measuring the release of glycerol from rat epididymal fat maintained in the presence of dexamethasone (0.2 microgram/ml) and the material to be tested (0.1 and 0.2 mg/ml). Although two preparations of pituitary bGH stimulated a significant (P less than 0.01) increase in glycerol production, neither rebGH nor recombinant-DNA-derived chicken GH was lipolytic. However, when rebGH was intravenously injected into three sheep (0.15 mg/kg), the increase in plasma nonesterified fatty acids was similar to that measured with the same dose of pituitary bGH. Insulin-tolerance tests were conducted in sheep before and after treatment with rebGH and pituitary bGH (four subcutaneous injections of 0.15 mg/kg). Although the effect of rebGH was less than that of the pituitary hormone, both significantly impaired the ability of insulin to lower the concentration of plasma glucose. These data suggest that the lipolytic and diabetogenic activities of bGH are intrinsic properties of the molecule. However, the lipolytic activity may only become apparent after either modification of the molecule in vivo or activation of a lipolytic intermediate.

Project description:Biomarker profiling, as a rapid screening approach for detection of hormone abuse, requires well selected candidate biomarkers and a thorough in vivo biomarker evaluation as previously done for detection of growth hormone doping in athletes. The bovine equivalent of growth hormone, called recombinant bovine somatotropin (rbST) is (il)legally administered to enhance milk production in dairy cows. In this study, first a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA) were developed for simultaneous measurement of four candidate biomarkers selected from literature: insulin-like growth factor 1 (IGF-1), its binding protein 2 (IGFBP2), osteocalcin and endogenously produced antibodies against rbST. Next, bovine serum samples from two extensive controlled rbST animal treatment studies were used for in vivo validation and biomarker evaluation. Finally, advanced statistic tools were tested for the assessment of biomarker combination quality aiming to correctly identify rbST-treated animals. The statistical prediction tool k-nearest neighbours using a combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12% of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. From the results of this multidisciplinary study, it is concluded that the osteocalcin - anti-rbST-antibodies combination represent fit-for-purpose biomarkers for screening of rbST abuse in dairy cattle and can be reliably measured in both the developed 4-plex FCIA as well as in a cost-effective 2-plex microsphere-based binding assay. This screening method can be incorporated in routine veterinary monitoring programmes: in the European Union for detection of rbST abuse and in the control of rbST-free dairy farms in the United States of America and other countries.

Project description:The present study evaluated the growth and puberty attainment of Bos indicus heifers administered recombinant bovine somatotropin (bST) or saline injections during preweaning and/or postweaning. On day 0, 177 suckling Nellore heifers were stratified by initial age and body weight (BW) (80 ± 10 d; 97 ± 16 kg), and randomly assigned, in a 2 × 2 factorial design (n = 44 to 45 heifers/treatment), to receive s.c. injections of saline (5 mL 0.9% NaCl) or sometribove zinc (Posilac; Elanco, Greenfield, IN; 6.14 mg/kg of BW0.75) on days 0 and 10 (PRE) and/or days 167 and 177 (POS). All heifers were managed as a single group in Brachiaria decumbens pastures from day 0 until 24 d postweaning (day 191), and then provided a corn silage-based TMR from days 191 to 380 to achieve 65% to 70% of mature BW at the end of the study (day 380). Heifer full BW was collected on days 0, 10, 167, 177, and monthly from days 191 to 380. Transrectal ultrasonography of ovaries was performed on days 1 and 10 of each month from days 229 to 380 to assess the percentage of pubertal heifers. Liver biopsies and blood samples from jugular vein were collected on days 0, 10, 167, 177, and 380. Additional blood samples were collected monthly from days 259 to 380 (n = 10 to 15 heifers/treatment). No interactions among day of the study, PRE, and POS injections of saline or bST were detected (P ? 0.11). Preweaning bST injections increased heifer average daily gain (ADG) from days 0 to 10 and plasma IGF-1 on day 10 (P ? 0.03), did not affect ADG from days 0 to 177, plasma IGF-1 from days 259 to 380, and any liver gene mRNA expression (P ? 0.19), but tended to decrease ADG from days 191 to 380 (P = 0.07) and percentage of pubertal heifers on days 349 (P = 0.07), 359 (P = 0.002), and 380 (P = 0.0001) compared with saline injections. Postweaning bST injections increased plasma IGF-1 on day 177 and overall liver mRNA expression of GHR-1A (P ? 0.05), decreased plasma IGF-1 from days 259 to 380 (P = 0.03), tended to decrease liver mRNA expression of GHR-1B on day 177 (P = 0.08), but did not affect ADG from days 167 to 177 and 191 to 380, and puberty attainment from days 229 to 380 (P ? 0.12) compared with saline injections. Thus, preweaning and postweaning injections of bST successfully increased heifer plasma IGF-1 concentrations 10 d after first injection. Postweaning injections of bST had no impact on puberty attainment, whereas preweaning bST injections of bST impaired puberty attainment of Nellore beef heifers.

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Covalent cross-linking of the bovine somatotropin dimer. Effects on growth-promoting, receptor-binding and immunological activities and preliminary characterization of the self-association.

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