An interchain disulfide dimer of human growth hormone

U. J. Lewis, S. M. Peterson, Lynda Bonewald, B. K. Seavey, W. P. VanderLaan

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Abstract

Human pituitary glands contained a form of growth hormone with a molecular weight of 45,000 that did not dissociate in 8 M urea or 1% sodium dodecyl sulfate. The material was isolated by a combination of ion exchange chromatography on DEAE-cellulose in a urea-containing buffer and gel filtration on Sephadex. Upon reduction with 2-mercapto-ethanol or oxidation with performic acid prior to electrophoresis in sodium dodecyl sulfate, the 45,000-dalton protein was converted into material with a molecular weight of 22,000. The 45,000-dalton form had a low growth-promoting activity, 0.14 IU/mg, and, after reduction and Cysteine-carbamidomethylation, the potency of the material increased to 0.55 IU/mg. The lactogenic activity of the 45,000 dalton form was 1.7 IU/mg and this remained essentially unchanged upon reduction and alkylation. In a radioimmunoassay the 45,000-dalton protein was about 50% and the 22,000-dalton Cysteine carbamidomethylated material, about 35% as effective as human growth hormone in displacing labeled growth hormone. Phenylalanine was the NH 2-terminal residue for the 45,000-dalton form and for the Cysteine-carbamidomethylated 22,000-dalton material. Sequence analysis by the Edman procedure indicated the first 6 residues of these materials to be the same as those found in human growth hormone. Phenylalanine was the COOH-terminal amino acid and glycine the penultimate residue. Formation of the 45,000-dalton protein was not detected after permitting monomeric growth hormone to stand at pH 9 for 1 week. Since all purification steps of the 45,000-dalton material were carried out at a pH of between 6.5 and 8.3, its formation by disulfide interchange seemed unlikely. We concluded that the 45,000-dalton protein was a dimer of growth hormone formed by interchain disulfide bonding. In addition to the disulfide dimer, another dimeric form of human growth hormone was detected which was resistant to reduction by 2-mercaptoethanol. The substance was only about 15% as active as human growth hormone in a radioimmunoassay, but end group analyses indicated the same termini as found in human growth hormone.

Original languageEnglish (US)
Pages (from-to)3697-3702
Number of pages6
JournalJournal of Biological Chemistry
Volume252
Issue number11
StatePublished - 1977
Externally publishedYes

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Human Growth Hormone
Disulfides
Dimers
Growth Hormone
Cysteine
Mercaptoethanol
Phenylalanine
Sodium Dodecyl Sulfate
Radioimmunoassay
Urea
Proteins
Molecular Weight
DEAE-Cellulose
Ion Exchange Chromatography
Alkylation
Molecular weight
Pituitary Gland
Glycine
Gel Chromatography
Sequence Analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Lewis, U. J., Peterson, S. M., Bonewald, L., Seavey, B. K., & VanderLaan, W. P. (1977). An interchain disulfide dimer of human growth hormone. Journal of Biological Chemistry, 252(11), 3697-3702.

An interchain disulfide dimer of human growth hormone. / Lewis, U. J.; Peterson, S. M.; Bonewald, Lynda; Seavey, B. K.; VanderLaan, W. P.

In: Journal of Biological Chemistry, Vol. 252, No. 11, 1977, p. 3697-3702.

Research output: Contribution to journalArticle

Lewis, UJ, Peterson, SM, Bonewald, L, Seavey, BK & VanderLaan, WP 1977, 'An interchain disulfide dimer of human growth hormone', Journal of Biological Chemistry, vol. 252, no. 11, pp. 3697-3702.
Lewis UJ, Peterson SM, Bonewald L, Seavey BK, VanderLaan WP. An interchain disulfide dimer of human growth hormone. Journal of Biological Chemistry. 1977;252(11):3697-3702.
Lewis, U. J. ; Peterson, S. M. ; Bonewald, Lynda ; Seavey, B. K. ; VanderLaan, W. P. / An interchain disulfide dimer of human growth hormone. In: Journal of Biological Chemistry. 1977 ; Vol. 252, No. 11. pp. 3697-3702.
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abstract = "Human pituitary glands contained a form of growth hormone with a molecular weight of 45,000 that did not dissociate in 8 M urea or 1{\%} sodium dodecyl sulfate. The material was isolated by a combination of ion exchange chromatography on DEAE-cellulose in a urea-containing buffer and gel filtration on Sephadex. Upon reduction with 2-mercapto-ethanol or oxidation with performic acid prior to electrophoresis in sodium dodecyl sulfate, the 45,000-dalton protein was converted into material with a molecular weight of 22,000. The 45,000-dalton form had a low growth-promoting activity, 0.14 IU/mg, and, after reduction and Cysteine-carbamidomethylation, the potency of the material increased to 0.55 IU/mg. The lactogenic activity of the 45,000 dalton form was 1.7 IU/mg and this remained essentially unchanged upon reduction and alkylation. In a radioimmunoassay the 45,000-dalton protein was about 50{\%} and the 22,000-dalton Cysteine carbamidomethylated material, about 35{\%} as effective as human growth hormone in displacing labeled growth hormone. Phenylalanine was the NH 2-terminal residue for the 45,000-dalton form and for the Cysteine-carbamidomethylated 22,000-dalton material. Sequence analysis by the Edman procedure indicated the first 6 residues of these materials to be the same as those found in human growth hormone. Phenylalanine was the COOH-terminal amino acid and glycine the penultimate residue. Formation of the 45,000-dalton protein was not detected after permitting monomeric growth hormone to stand at pH 9 for 1 week. Since all purification steps of the 45,000-dalton material were carried out at a pH of between 6.5 and 8.3, its formation by disulfide interchange seemed unlikely. We concluded that the 45,000-dalton protein was a dimer of growth hormone formed by interchain disulfide bonding. In addition to the disulfide dimer, another dimeric form of human growth hormone was detected which was resistant to reduction by 2-mercaptoethanol. The substance was only about 15{\%} as active as human growth hormone in a radioimmunoassay, but end group analyses indicated the same termini as found in human growth hormone.",
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AU - VanderLaan, W. P.

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N2 - Human pituitary glands contained a form of growth hormone with a molecular weight of 45,000 that did not dissociate in 8 M urea or 1% sodium dodecyl sulfate. The material was isolated by a combination of ion exchange chromatography on DEAE-cellulose in a urea-containing buffer and gel filtration on Sephadex. Upon reduction with 2-mercapto-ethanol or oxidation with performic acid prior to electrophoresis in sodium dodecyl sulfate, the 45,000-dalton protein was converted into material with a molecular weight of 22,000. The 45,000-dalton form had a low growth-promoting activity, 0.14 IU/mg, and, after reduction and Cysteine-carbamidomethylation, the potency of the material increased to 0.55 IU/mg. The lactogenic activity of the 45,000 dalton form was 1.7 IU/mg and this remained essentially unchanged upon reduction and alkylation. In a radioimmunoassay the 45,000-dalton protein was about 50% and the 22,000-dalton Cysteine carbamidomethylated material, about 35% as effective as human growth hormone in displacing labeled growth hormone. Phenylalanine was the NH 2-terminal residue for the 45,000-dalton form and for the Cysteine-carbamidomethylated 22,000-dalton material. Sequence analysis by the Edman procedure indicated the first 6 residues of these materials to be the same as those found in human growth hormone. Phenylalanine was the COOH-terminal amino acid and glycine the penultimate residue. Formation of the 45,000-dalton protein was not detected after permitting monomeric growth hormone to stand at pH 9 for 1 week. Since all purification steps of the 45,000-dalton material were carried out at a pH of between 6.5 and 8.3, its formation by disulfide interchange seemed unlikely. We concluded that the 45,000-dalton protein was a dimer of growth hormone formed by interchain disulfide bonding. In addition to the disulfide dimer, another dimeric form of human growth hormone was detected which was resistant to reduction by 2-mercaptoethanol. The substance was only about 15% as active as human growth hormone in a radioimmunoassay, but end group analyses indicated the same termini as found in human growth hormone.

AB - Human pituitary glands contained a form of growth hormone with a molecular weight of 45,000 that did not dissociate in 8 M urea or 1% sodium dodecyl sulfate. The material was isolated by a combination of ion exchange chromatography on DEAE-cellulose in a urea-containing buffer and gel filtration on Sephadex. Upon reduction with 2-mercapto-ethanol or oxidation with performic acid prior to electrophoresis in sodium dodecyl sulfate, the 45,000-dalton protein was converted into material with a molecular weight of 22,000. The 45,000-dalton form had a low growth-promoting activity, 0.14 IU/mg, and, after reduction and Cysteine-carbamidomethylation, the potency of the material increased to 0.55 IU/mg. The lactogenic activity of the 45,000 dalton form was 1.7 IU/mg and this remained essentially unchanged upon reduction and alkylation. In a radioimmunoassay the 45,000-dalton protein was about 50% and the 22,000-dalton Cysteine carbamidomethylated material, about 35% as effective as human growth hormone in displacing labeled growth hormone. Phenylalanine was the NH 2-terminal residue for the 45,000-dalton form and for the Cysteine-carbamidomethylated 22,000-dalton material. Sequence analysis by the Edman procedure indicated the first 6 residues of these materials to be the same as those found in human growth hormone. Phenylalanine was the COOH-terminal amino acid and glycine the penultimate residue. Formation of the 45,000-dalton protein was not detected after permitting monomeric growth hormone to stand at pH 9 for 1 week. Since all purification steps of the 45,000-dalton material were carried out at a pH of between 6.5 and 8.3, its formation by disulfide interchange seemed unlikely. We concluded that the 45,000-dalton protein was a dimer of growth hormone formed by interchain disulfide bonding. In addition to the disulfide dimer, another dimeric form of human growth hormone was detected which was resistant to reduction by 2-mercaptoethanol. The substance was only about 15% as active as human growth hormone in a radioimmunoassay, but end group analyses indicated the same termini as found in human growth hormone.

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