Human pituitary growth hormone. XXI. Physicochemical investigations of the native and reduced-alkylated protein

Bewley, Thomas A.; Brovetto-Cruz, Jorge; Li, Choh Hao

doi:10.1021/bi00840a007

Cited by 143 publications

(102 citation statements)

References 17 publications

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“…Our assay data on the completely reduced and alkylated HGH derivatives confirm the earlier results of Li and coworkers [1][2][3][4] : complete reduction and carboxymethylation of both disulfide bonds in HGH abolishes the growth promoting activity of the hormone [3], whereas reduction and carbamidomethylation of the disulfide bridges do not alter the biological properties of HGH [1][2][3][4]. The slight biological activity of tetra-S-carboxymethylated HGH prepared according to Bewley et al [2] (table 2) may be due to the presence of a small amount of contaminating di-S(182,189)-carboxymethylated HGH in this preparation (see tube f in fig.3).…”

Section: Discussionsupporting

confidence: 90%

“…Different methods have been reported for complete reduction and alkylation of the two disulfide bonds in the structure of human growth hormone (HGH) [1][2][3][4]. More recent studies on partial reduction and alkylation of the disulfide bonds in porcine [5], human [6] and bovine growth hormones [7] indicated that the small disulfide loop near the COOHterminus in these hormones is much more exposed to reducing agents than the large loop.…”

Section: Introductionmentioning

confidence: 99%

“…More recent studies on partial reduction and alkylation of the disulfide bonds in porcine [5], human [6] and bovine growth hormones [7] indicated that the small disulfide loop near the COOHterminus in these hormones is much more exposed to reducing agents than the large loop. It has been reported that the completely reduced and carbamidomethylated derivative of HGH retains the full biological potency of the native hormone [1][2][3][4], whereas complete reduction and carboxymethylation of the disulfide bonds in HGH leads to a considerable loss of the biological activities [4]. In view of this latter observation it has been of interest to attempt the selective reduction and carboxymethylation of the COOH-terminal disulfide loop of HGH, and to determine the effect of this modification on the biological properties of HGH.…”

Section: Introductionmentioning

confidence: 99%

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

Gráf¹,

Borvendég²,

Barát³

et al. 1976

FEBS Letters

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Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Gráf¹,

Borvendég²,

Barát³

et al. 1976

FEBS Letters

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Section: Methodsmentioning

confidence: 99%

“…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).…”

Section: Methodsmentioning

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.

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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...

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The Chemistry of Human Pituitary Growth Hormone

Bewley¹,

Li²

1975

Advances in Enzymology - And Related Areas of Molecular Biology

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Human pituitary growth hormone. XXI. Physicochemical investigations of the native and reduced-alkylated protein

Cited by 143 publications

References 17 publications

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

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.

The Chemistry of Human Pituitary Growth Hormone

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