We have undertaken studies to determine the effect of glycosylation on the lactogenic activity of ovine PRL (oPRL). Measuring casein production in the in vitro mouse mammary gland explant assay, we found that glycosylated oPRL had 80% of the activity of oPRL. In competitive binding studies using lactogen receptors from mammary glands of lactating rabbits, glycosylated oPRL had only 20% the potency of oPRL. In the Nb2 assay also, glycosylated oPRL was approximately 24% as potent as oPRL in stimulating mitogenic activity. Thus, these studies show that the glycosylated variant of PRL has less biological activity than the major PRL form and that the alteration of an activity by glycosylation is selective.

PITUITARY dwarfism may be due to a deficiency of growth hormone or a lack of peripheral action of growth hormone. Lack of peripheral action of growth hormone may be secondary to an abnormality in the structure of the hormone,1 an inability of growth hormone to generate growth-promoting substances, such as the somatomedins or insulin-like growth factors,2 3 4 or nonresponsiveness of target tissue to these growth-promoting factors.5 The nonresponsiveness of target tissue to growth factors may be due to a defect in the cell-membrane receptors or to any step beyond receptor binding.Although growth hormone deficiency is easily identified by low plasma . . .

The intra-lymph node technique used to inoculate rabbits with small quantities of antigen has been described. A variety of antigens in the 20,000-22,000 molecular weight range, as well as a 15-amino acid peptide coupled to BSA, have been inoculated successfully by this procedure. We have made no attempt to compare the success rate of the intra-lymph node inoculation route with other techniques utilizing small (microgram) quantities of antigen.

A radioimmunoassay for the 15-amino acid fragment comprising residues 32–46 of the 22,000-dalton human growth hormone has been devised. The radioimmunoassay detects from 1 to 150 fmol of the synthetic peptide. The 12-amino acid peptide hGH (35–46) shows parallel displacement, but the sensitivity decreased 50% due to the deletion of the three amino acids. Displacement activity is lost from the synthetic peptides hGH (38–46) and hGH (43–46), and intact hGH itself shows no displacement. Disrupting the large loop of hGH by subtilisin cleavage or by reduction and alkylation of the S-S bridges imparts displacement activity parallel to that of hGH (32–46), and hGH (1–139) has similar activity. A plausible interpretation for these results is that native hGH has a tertiary structure that masks or obscures the sequence 32–46, the sequence being unmasked by release of constraints imposed by an intact large loop.

A 13 amino acid analog of the human prolactin amino terminus was synthesized, substituting tyrosine for valine at residue 13. The peptide was coupled to crystalline bovine serum albumin for antisera production. The peptide was used for iodination with 125I, and displacement curves were found to be parallel when human prolactin and the synthetic peptide were compared as standards. The radioimmunoassay using the synthetic peptide has the advantages of purity in its roles as hapten in the antigen and as labelled peptide, of ease of iodination of the peptide, of its stability after iodination, and of obviating the need for native human prolactin. The radioimmunoassay is suitable for the measurement of human prolactin concentration in plasma.

We compared the disappearance rate of hGH and hGH20K injected into groups of mice. Radioactivity measurements and RIA were used to determine the amount of hormone in blood collected at various times after injection. The results indicate that the T1/2 value for hGH and hGH20K are equal. We conclude that there is no apparent relationship between the equal rates of disappearance, the dissimilar binding characteristics that have been shown for the hormones and their equipotent growth promoting activities.

The dose-dependent displacement characteristics of a biologically active 20,000 molecular weight human pituitary growth hormone variant (20K) and of human growth hormone (hGH) were compared using hGH liver plasma membrane receptors both from 20-30 day pregnant rabbits and from normal female rats and also using mammary gland plasma membrane receptors from 5-6 day lactating rabbits. Different preparations of 20K were found to be only 3-20% as potent as hGH when compared at the dose necessary to cause 50% displacement of (125I)iodo-hGH from liver receptor and was 22-53% as effective as hGH in the mammary receptor assay system. These findings suggest that 20K and hGH may have separate receptors or that the binding characteristics of the two hormones may be quite different.

This chapter discusses the use of talc-resin trichloroacetic acid test for screening radioiodinated polypeptide hormones. The talc-resin TCA test is a rapid, reliable physicochemical method for predicting the immunologic performance of 125I-labeled hormones in radioimmunoassays (RIA). With the talc-resin TCA test, it is possible to anticipate the RIA behavior of 125I -labeled human (h) and rat (r) prolactin (PRL), growth hormone (GH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH) prepared from more than 100 iodinations. The talc-resin TCA test is based on three different physicochemical properties of iodohormones: adsorption to talc, exclusion from resin, and precipitation by trichloroacetic acid (TCA). The test patterns are distinct for monomeric iodohormone, aggregated iodohormone, and 125I-labeled salts. Thus, a usable iodohormone may be identified quickly and accurately by its meeting the criteria of greater than 90% talc adsorption, less than 25% bound to resin, and greater than 90% TCA precipitation. In addition, the talc and TCA percentages should agree within 3%. Radioiodinated human and rat PRL, TSH, GH, and LH have been prepared by different iodination procedures. All the purified monomeric iodohormones, producing sensitive and reliable RIA results, showed the characteristic talc-resin TCA test results of >90% talc adsorption, <25% resin binding, and >90% TCA precipitation.

A RIA system to measure two-chain forms of human GH (hGH) is described in this report. The need for such a RIA became evident with the identification of several single and two-chain peptides separated from pituitary hGH. hGH-S, a mixture of three peptides (des 140–149 and des 140–146 with residue 152 either Asn or Asp) was produced by subtilisin cleavage of hGH and provided a basis for development of the RIA of two-chain forms of hGH; in this RIA, two-chain forms were used as antigen, for iodination, and for standards. Competitive binding-displacement characteristics of several naturally occurring single chain and two-chain forms of hGH were assessed in the hGH-S RIA system and in a standardized hGH RIA using materials supplied by NIAMDD. In addition, enzymically produced two-chain forms were tested. These studies show that the antiserum used in the hGH RIA reacts to varying degrees with both single and two-chain forms of GH, whereas the antiserum to hGH-S reacts only with some forms of two-chain hGH and their N-terminal fragments (F1), obtained by reduction of the disulfide bridges, followed by alkylation of the two-chain forms of hGH. The hGH-S RIA may be useful for the measurement of twochain forms of hGH found in pituitary extracts as well as in blood. Specific assays such as this may provide a means to resolve some of the contradictory data seen when results from RIAs are compared with results from either bioassays or radioreceptor assays. (Endocrinology106: 92, 1980)

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.

The talc-resin-TCA test is a rapid, reliable method for predicting the behavior of 125I-hormones in radioimmunoassays (RIA). This test is based on the assessment of three different physicochemical properties of iodinated hormones: adsorption to talc, exclusion from ion exchange resin, and precipitation by TCA. These three parameters produce test patterns that are unique for monomeric 125I-hormone, aggregated 125I-hormone, and 125I-salts. Different iodination methods were used to prepare radioiodinated human and rat PRL, GH, TSH, and LH, and rat FSH. Purified monomeric 125I-hormones that gave reliable RIA results all showed a characteristic talc-resin-TCA profile of >90% talc adsorption, <25% resin binding, and >90% TCA precipitation. This profile has successfully predicted reliable RIA results for 125I-hormones prepared from more than 80 iodinations.