Abstract
Extracts of frozen human pituitary glands contain, in addition to normal growth hormone (hGH (Mr = 22,005), a variant of lower molecular weight (approximately 20,000) (Singh, R. N. P., Seavey, B. K. and Lewis, U. J. (1974) Endocrinol. Res. Commun. 1, 449-464). This material, designated 20K hGH, occurs in various forms with the majority present as a heterologous dimer of Mr = 20,000 hGH and hGH. The purification scheme for obtaining the variant from these fractions is described. Analysis of Mr = 20,000 hGH in the sequenator has confirmed that residues 32-46 of hGH are absent in Mr = 20,000 hGH. The structures of hGH and Mr = 20,000 hGH have been compared by 1H nuclear magnetic resonance spectroscopy, circular dichroism, spectrophotometric titration, and nitration of the tyrosine residues by tetranitromethane. The results indicate that the folding of the polypeptide chain of the two proteins is similar but not identical.
Highlights
PREPARATION AND SOME PHYSICAL AND CHEMICAL PROPERTIES*The to remove a resulting solution was centrifuged at 16,500 X g for 2 small amount of undissolved material, and the supergrowth hormone in extracts of human pituitary glands was natant was applied to a column of Sephadex GI00 (10 X 90 cm)
From the +NationaHl ormone Laboratory, Department ofBiochemistry, University of Auckland, Private Bag, Auckland, New Zealand, theTDepartment ofBiological Chemistry, Washington University School of Medicine, St
We have investigated the forms in which the M, = 20,000 (20K) variant occurs in extracts of human pituitary glands and have devised methods for purifying it
Summary
The to remove a resulting solution was centrifuged at 16,500 X g for 2 small amount of undissolved material, and the supergrowth hormone in extracts of human pituitary glands was natant was applied to a column of Sephadex GI00 (10 X 90 cm). It fist reportedby Singh et al (1). Subsequent work showed was eluted at 130 ml/h with 50 mM Tris/acetic acid, pH 8.5 The this variant to be a single chain polypeptide with NH2- and absorbance of the column effluent at 280nm was monitored and COOH-terminal sequences identical with those of hGH' and fractions containing hGH dimer were pooled for further processing.
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