BIOASSAY OF HYPOPHYSEAL GROWTH HORMONE: THE TIBIA TEST<sup>1</sup>

“…The growth promoting and prolactin activities were determined by the rat tibia test [10] and the quantitative pigeon crop-sac assay [11 ], respectively. Table 1 presents the course of reduction and alkylation with increasing amounts of dithiothreitol in the presence and in the absence of 8 M urea.…”

Section: Preparation and Characterization Of Reduced Tetra-s-and Di-smentioning

confidence: 99%

Reactivity and biological importance of the disulfide bonds in human growth hormone

Gráf¹,

Borvendég²,

Barát³

et al. 1976

FEBS Letters

View full text Add to dashboard Cite

“…Highly purified HGH (3) was used for the preparation of standards and labeling with '25I essentially as described (11). The growth-promoting activity was determined by the rat tibia test (12). Fig.…”

mentioning

confidence: 99%

Human somatotropin: restoration of full biological activity by noncovalent interaction of a natural and a synthetic fragment of the hormone.

Li¹,

Bewley²,

Blake³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The recombinant hormone obtained by noncovalent interaction of the natural NH2-terminal fragment (consisting of 134 amino acid residues) with a synthetic COOH-terminal fragment of 52 amino acids of the reducedcarbamidomethylated human somatotropin molecule is found to exhibit full biological activity of the native hormone as evidenced by the tibia test. Radioimmunoassay data indicate that the semisynthetic recombinant hormone possesses essentially full immunoreactivity as compared to the native one. In addition, circular dichroism and fluorescence emission spectra of the semisynthetic recombinant are identical to that of the undissociated, plasmin modified, reduced-carbamidomethylated hormone.Restoration of full biological activity by noncovalent interaction of the NH2-terminal fragment [consisting of 134 amino acid residues from the reduced-carbamidomethylated human somatotropin (HGH) (see Fig. 1)] with the COOH-terminal fragment of 51 amino acids has recently been described (1, 2). In this paper, we report complementation experiments using the natural Cys(Cam)53-HGH-(1-134) and the synthetic Cys-(Cam) 'C35, to obtain a recombinant hormone with full growth-promoting and immunochemical potency. MATERIALS AND METHODSHuman somatotropin was isolated from fresh-frozen pituitary glands by the method of Li et al. (3). Plasmin digestion of HGH and the isolation of Cys(Cam)53-HGH-(1-134) were carried out as described previously (4). The NH2-terminal fragment was further purified by gel filtration on Sephadex G-100 in 0.01 M NH4HCO3 at pH 8.2 (5). The COOH-terminal fragment, Cys(Cam) 165,182, was synthesized as described (6). The complementation experiments were carried out by the recently published procedure (1). Exclusion chromatography of the reaction mixture was performed at pH 8.2 in 0.1 M Tris-HCI buffer containing 0.2% NaN3 on Sephadex G-100. Rechromatography was done after concentration of the fraction by ultrafiltration (Amicon PM-10 membrane).Circular dichroism (CD) spectra were taken on a Cary model 60 spectropolarimeter equipped with a model 6002 circular dichroism attachment according to procedures outlined previously (7). The a-helix content was estimated as described by Bewley et al. (8). The concentration of recombinant hormone was determined spectrophotometrically by using the absorptivity value previously reported for native HGH (8). Fluorescence emission spectra were taken on a Hitachi Perkin Elmer spectrofluorimeter, model MPF-2A, according to procedures described elsewhere (9). The excitation monochrometer was set at 294 nm in all cases.For radioimmunoassay, the double-antibody procedure of Schalch and Reichlin (10) was used with slight modifications, by employing a guinea pig antiserum against human somatotropin. Highly purified HGH (3) was used for the preparation of standards and labeling with '25I essentially as described (11). The growth-promoting activity was determined by the rat tibia test (12). Fig. 2A shows the elution pattern of the reaction mixture on Sephadex G-100. Approximate...

show abstract

BIOASSAY OF HYPOPHYSEAL GROWTH HORMONE: THE TIBIA TEST¹

Cited by 388 publications

References 0 publications

The effect of growth hormone treatment on growth in zinc deficient rats

The effect of growth hormone treatment on growth in zinc deficient rats

Reactivity and biological importance of the disulfide bonds in human growth hormone

Human somatotropin: restoration of full biological activity by noncovalent interaction of a natural and a synthetic fragment of the hormone.

Contact Info

Product

Resources

About

BIOASSAY OF HYPOPHYSEAL GROWTH HORMONE: THE TIBIA TEST1

Cited by 388 publications

References 0 publications

The effect of growth hormone treatment on growth in zinc deficient rats

The effect of growth hormone treatment on growth in zinc deficient rats

Reactivity and biological importance of the disulfide bonds in human growth hormone

Human somatotropin: restoration of full biological activity by noncovalent interaction of a natural and a synthetic fragment of the hormone.

Contact Info

Product

Resources

About

BIOASSAY OF HYPOPHYSEAL GROWTH HORMONE: THE TIBIA TEST¹