R. N. P. Singh

Human Growth Hormone: A Complex of Proteins

Publisher Summary This chapter discusses that the human growth hormone is a complex of proteins. Human growth hormone stands apart from all other pituitary hormones when one considers the amount present in the gland. It amounts to 8 mg/gland as compared to the other hormones which are found in microgram amounts. Using analytical and preparative techniques, it has been found that growth hormone is not a single substance but a mixture of variants differing in amino acid sequence, posttranslational modified forms, and fragments. The chapter focuses on the experimental results that support the concept that each of the forms can be a separate hormone with a different physiological role and only when taken altogether can the mixture account for the complex and often contradictory actions of the hormone. The concept that growth hormone is a mixture of different forms is stimulated by two reports—the elegant analytical gel electrophoresis technique, and that growth hormone is less effective in inhibiting glucose uptake if recrystallized. The electrophoretic approach makes it apparent that preparations of growth hormone are not only heterogeneous, but also the hormone is contaminated with a proteinase that readily produces hydrolytic products that contributes to the heterogenity.

Human prolactin: Isolation and some properties

Summary A protein with a potency of 22 IU/mg in the pigeon crop sac assay for prolactin but only 0.4 USP U/mg in the tibial line assay for growth hormone has been isolated from pooled human pituitary glands. When tested by radioimmunoassay for human growth hormone, 12 ng of the material was equivalent to 1 ng of a reference growth hormone. The protein was less electronegative than growth hormone at pH 9.5 and had a molecular weight of about 22,000.

Heterogeneity of Human Growth Hormone

Purified preparations of human growth hormone (hGH) consistently showed two growth hormone-like contaminants when analyzed by SDS-gel electrophoresis (SDS — sodium dodecyl sulfate). Because of their behavior during SDS-electrophoresis, the two substances have been designated as 24K and 20K. This distinguishes them from hGH with a molecular weight of 22,100 (22K). The substances were difficult to detect by disc electrophoresis (pH 10) because they migrated with nearly the same mobility as hGH. Separation of the contaminants from hGH was accomplished by chromatography on Sephadex G-75 with 6 M urea. A dimer of hGH which was stable to urea and SDS but which dissociated with mercaptoethanol also was purified during these studies.The 24K form was hGH with a single nick in the large disulfide loop. This cleavage altered the conformation of the molecule such that the effective radius of the protein was increased and Its migration slowed during gel electrophoresis. The 20K modification had no cleavage in the larg...

Bovine growth hormone: Evidence for two allelic forms

Summary Peptide maps of tryptic digests of bovine growth hormone isolated from individual pituitary glands were of three types. The hormone contained either an A or a B peptide or a 1:1 mixture of the two. A and B differed by a substitution of valine for leucine. The only difference noted between the ovine and bovine growth hormones was that the ovine hormone had neither A nor B but instead a peptide that differed from A by a replacement of a glycine with valine.

Human growth hormone peptide 1–43: Isolation from pituitary glands

A procedure is described for isolation from human pituitary glands of a peptide with the amino acid sequence of the first 43 residues of human growth hormone, hGH (1–43). The peptide was recovered in a yield of 12 mg per 1000 pituitary glands. In a radioimmunoassay for hGH the peptide was unreactive when tested at 1 µg/ml and therefore if it does circulate, it probably has gone underected in blood. Growth-promoting activity as measured by the tibial line procedure was low but detectable; however, the log dose response was not parallel to that of hGH. Separate studies indicated the hGH (1–43) was a potent potentiator of insulin action suggesting physiologic significance. Further, because the peptide can be a cleavage product of the major form of hGH but not of its 20,000-dalton variant, importance is indicated for the multiple forms of the hormone.

Enhancement of the growth promoting activity of human growth hormone.

Abstract The growth promoting activity of human growth hormone was enhanced at least 4 to 5-fold by controlled digestion with a bacterial proteinase. The increased growth activity was measured in rats by both the tibial line and weight gain assays. By radioimmunoassay the potentiated material was indistinguishable from intact human growth hormone. Pigeon crop sac stimulating activity was increased 4 to 10-fold by the enzymic modification. Three forms denoted as I, II and III were formed during the digestion. These were separated by chromatography on DEAE-cellulose and all were similar in enhanced biological potencies. Peptide mapping indicated the three forms lacked residues 138–147 but other structural differences could not be determined by this technique. The activation process involved, therefore, conversion of the hormone from a single chain to a double chain-structure.

Hyperglycemic Activity of the 20,000-Dalton Variant of Human Growth Hormone

The 20,000-dalton structural variant of human growth hormone was inactive as a hyperglycemic agent in dogs when injected 10 h prior to a glucose tolerance test. Limited digestion with subtilisin did not generate hyperglycemic activity. These results are in contrast to those obtained with human growth hormone where subtilisin treatment potentiated the weak hyperglycemic activity of the undigested hormone. The results suggest that the 15 amino acid sequence that is deleted from the variant is either directly responsible for hyperglycemic activity or that a modification in tertiary structure produced by the deletion prevents a necessary proteolytic processing.

Comparison of the tryptic peptides of human, porcine and ovine prolactins

Abstract Tryptic peptides of ovine, porcine, and human prolactins were separated by two-dimensional chromatography-electrophoresis, and the amino acid composition of each peptide was determined. Similarities in the compositions of the peptides from the three species permitted tentative alignments of the amino acids of the porcine and human peptides with identical or similar residues of the ovine hormone. If the suggested sequences are correct, the peptides of human prolactin were more like those of the nonprimate prolactins than those of human growth hormone.

Procedure for isolation of the 20,000-dalton variant of human growth hormone.

An isolation procedure for the 20,000-dalton variant of human growth hormone has been devised to improve the yield of the final product. The improvement involved elimination of cumbersome steps that decreased yield, and modification of chromatography on DEAE-cellulose to provide better separation of the variant from the major form of growth hormone.