U. J. Lewis

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.

The 20,000-dalton variant of human growth hormone: Location of the amino acid deletions☆

Abstract The 20,000-dalton variant of human growth hormone lacks a sequence of 15 amino acids normally found in the hormone. The deletion involves residues 32 through 46. The remainder of the molecule is identical to the larger form of the hormone. These results suggest that the variant is the product of a deletion mutation in the growth hormone gene.

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.

Kinetic study of the deamidation of growth hormone and prolactin

Abstract 1. 1.|Pituitary growth hormore and prolactin are converted to more acidic electrophoretic components when they are allowed to stand in an alkaline medium. A quantitative study of this instability was made by measuring the first-order rate constant for the conversion of the major electrophoretic component to the faster-migrating bands. Changes in concentration of the components were determined by colorimetric assay of the stained disc-electrophoretic bands. 2. 2.|An increase in temperature, pH or ionic strength of the solution, or use of urea, increased the rate of alteration of bovine growth hormone, ovine prolactin and human growth hormone. Urea not only accelerated the conversion, but also increased the number of converted forms. 3. 3.|The rate constants for the conversion were greatest for ovine prolactin under all conditions. Human growth hormone was generally more stable to alteration than ovine prolactin. Bovine growth hormone was the least susceptible to conversion. 4. 4.|The rate of liberation of NH 3 from the hormones at pH 10 correlated with the rate of conversion of the major electrophoretic component. The data were consistent with the assumption that each faster-migrating component was formed by loss of one mole of NH 3 . 5. 5.|The multiple disc-electrophoretic forms of ovine prolactin were analyzed by isoelectric focusing in carrier ampholytes. The principal band behaved as a single component, whereas each of the faster-migrating bands was resolved into at least two components. 6. 6.|It was concluded that increased electrophoretic heterogeneity of the hormones, in the absence of proteinases, can be attributed to a nonenzymic deamidation.

The 20,000-dalton structural variant of human growth hormone: Lack of some early insulin-like effects☆

In contrast to the major form of human growth hormone the 20,000-dalton (20K) variant of the hormone produced no decrease in either serum glucose of free fatty acids one hour after injection into fasted, hypophysectomized rats. Furthermore, the variant caused no rise in serum free fatty acids after 5 hours. Invitro experiments utilizing epididymal adipose tissue from hypophysectomized rats indicated that 20K was unable to accelerate glucose utilization as measured by glucose uptake and CO2 formation. The data show that this form, even though growth promoting, lacks some of the metabolic properties attributed to growth hormone. We conclude that an insulin-like effect is not necessarily a prerequisite for growth promoting activity.

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.

Effect of calcium, insulin and growth hormone on membrane fluidity. A spin label study of rat adipocyte and human erythrocyte ghosts.

ESR spectra were recorded from rat epididymal adipocyte ghosts labeled with the 5-nitroxide stearic acid spin probe, I(12,3). Polarity-corrected and approximate order parameters, that are sensitive to the flexibility of the incorporated label, were used to evaluate the membrane lipid fluidity. Addition of CaCl2 a 37 degrees C decreased the fluidity, as indicated by positive increases in the order parameters. The ordering effect of Ca2+ was concentration-dependent, reached saturation at approx. 3--4 mM, and was completely reversed by excess EGTA. Previous studies indicated that low- and high-affinity sites on adipocyte plasma membranes are able to bind 45Ca2+, and our results suggest that Ca2+-induced alterations in the lipid fluidity involve cation binding to low-affinity sites. The cellular movements of Ca2+ and, in particular, the binding of Ca2+ to the plasma membrane may play important roles in insulins action on fat cell function. The possibility that insulin directly alters the membrane fluidity was tested by adding hormone to freshly-prepared I(12,3)-labeled adipocyte ghosts. Insulin, at concentrations (10(-6) M) that enhance glucose uptake into intact adipocytes, did not affect the fluidity of ghosts suspended in buffers with or without Ca2+. The fluidities of I(12,3)-labeled rat adipocyte ghosts or human erythrocyte ghosts were also unaffected by various forms of human growth hormone.

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.