Form Of Human Growth Hormone Research Articles

Big growth hormone forms in human plasma: Immunochemical evidence for their pituitary origin

large molecular weight (oligomeric) forms of human growth hormone (hGH) are present in both pituitary extracts and plasma, but the origin of the plasma forms is not known. We used a monoclonal anti-hGH antibody (No. NA-71), previously employed to characterize hGH oligomers in pituitary extracts, to examine circulating hGH oligomers. This monoclonal antibody discriminated between monomeric and polymeric hGH forms in the pituitary. Plasma from normal subjects and acromegalic patients was fractionated by Sephadex G-100 chromatography and fractions were assayed by a monoclonal radioimmunoassay (RIA) using antibody NA-71 and by a polyclonal RIA. The oligomeric plasma hGH fraction showed decreased immunoreactivity in the monoclonal RIA, similar to that observed with pituitary oligomers. The immunopotency ratios of the various plasma hGH size isomers, as determined in the two RIAs, were virtually identical to those of pituitary hGH size isomers. We conclude that circulating hGH oligomers are immunochemically indistinguishable from pituitary hGH oligomers, and thus are most likely derived from pituitary stores.

Trypsin-resistant forms of human growth hormone have diabetogenic and insulin-like activities

Although diabetogenic and insulin-like activities are intrinsic properties of the growth hormone (GH) molecule, it has been frequently suggested that the hormone must be proteolytically processed for these activities to be expressed. If this is correct, then derivatives of GH having resistance to appropriate proteolytic attack might not have diabetogenic and/or insulin-like activity. The purpose of the present study was to prepare derivatives of human GH that are resistant to digestion by trypsin and to determine whether they posses diabetogenic or insulin-like activity. Three derivatives were prepared from purified native human GH in which lysine residues were modified with methyl acetimidate, citraconic anhhydride of S-ethylthioltrifluoroacetate, and one in which arginine residues were modified with camphorquinone-10-sulfonic acid. Comparisons of peptide maps of tryptic digests of these derivatives with that of unmodified human GH indicated that all four were resistant to proteolysis by trypsin. All of these trypsin-resistant forms of human GH were found to posses ssignificant growth-promoting, diabetogenic and insulin-like activities, although all activities were attenuated to some extent in each derivative. The relative potencies of the human GH derivatives in a radioimmunoassay for human GH were somewhat similar to their order of potency in the growth-promoting and diabetogenic assays. These results suggest that if proteolytic processing of the GH molecule is involved in the expression of one or more of its biological activities, such processing probably does not involve a trypsin-like proteinase.

Pseudomonas Aeruginosa Secretes and Correctly Processes Human Growth Hormone

Pseudomonas aeruginosa transformed with a plasmid containing a gene encoding the mature form of human growth hormone (hGH) preceded by an ATG codon to initiate translation expresses methionyl-hGH its cytoplasm. hGH produced in P. aeruginosa cells transformed with a plasmid containing a gene for the natural hGH precursor is transported across the inner membrane and has the NH2-terminal amino acid sequence of authentic mature hGH.

In vitro deletional mutagenesis for bacterial production of the 20,000-dalton form of human pituitary growth hormone.

The 20,000-dalton (20K) variant form of human growth hormone (hGH) present in extracts from pituitary glands differs from the major form of hGH (22K, 191 amino acids) by the deletion of amino acid residues 32-46. Using oligonucleotide-mediated mutagenesis, the DNA coding for these amino acids was deleted from the gene previously constructed by us (Goeddel et al., 1979) for microbial hGH production. The DNA to be deleted was looped out by the annealing of a synthetic oligodeoxyribonucleotide to the coding strand of the hGH gene contained on recombinant phage M13 mp8 DNA. Resulting heteroduplex structures were stabilized using primer-directed in vitro DNA synthesis in the presence of T4 DNA ligase. On transformation of Escherichia coli, these heteroduplex DNAs yielded phage whose genomes contained either the original or the partially deleted hGH gene, and genotypes were distinguished by in situ plaque hybridization with synthetic oligonucleotide probes. A gene with the correct deletion was used to express the short hGH variant in E. coli.

High-performance liquid chromatography of amino acids, peptides and proteins : XLVIII. Retention behaviour of tryptic peptides of human growth hormone isolated by reversed-phase high-performance liquid chromatography: A comparative study using different chromatographic conditions and predicted elution behaviour based on retention coefficients

The retention behaviour of the tryptic peptides of human growth hormone under a variety of reversed-phase chromatographic conditions has been investigated. By using water—acetonitrile gradients containing phosphate, bicarbonate, trifluoroacetate, or heptanesulphonate buffers, the influence of these ionic modifiers on peptide selectivity with several different alkylsilicas has been examined. Comparisons of observed and predicted retention behaviours for the various tryptic peptides in the different chromatographic systems are described. Application of these systems to the isolation of deamidated forms of human growth hormone is presented.

Antigenic, receptor-binding and mitogenic activity of proteolytic fragments of human growth hormone.

Analysis of the subtilisin-digested, two-chain form of human growth hormone (hGH) and its constituent polypeptide fragments has been aided by the use of monoclonal antibodies which bind specifically to four distinct epitopes on the native hormone. Using the SDS-polyacrylamide immunoblotting technique, it was shown that one epitope (shared with human chorionic somatomammotropin) detected by EB1 (or EB3) antibody was expressed to a similar extent by both the N-terminal (15 K) and C-terminal (7 K) polypeptides. This epitope is unique in that it represents a repeating determinant within the single chain structure of the hormone. Another three epitopes detected by monoclonal antibodies QA68/NA27, NA71 or NA39/EB2 were absent from the 7-K fragment but were expressed on the 15-K fragment to a similar extent to that on unmodified growth hormone. Binding of NA71 antibody was demonstrated only by radioimmunoassay since this, presumably conformational epitope could not be detected by immunoblotting. The functional importance of the 15-K peptide was demonstrated by its ability to bind specifically to hormone receptors on IM9 human lymphoblastoid cells and by its retention of mitogenicity for the NB2 rat lymphoma cell line. However, all tested monoclonal antibodies inhibited the binding of [125I]15-K to IM9 cell receptors by either steric hindrance or by an allosteric mechanism and therefore could not be further related topographically to the receptor-binding moiety of hGH.

Altered proteolytic cleavage of human growth hormone as a result of deamidation.

The major desamido form of human growth hormone (hGH) results from deamidation of asparagine 152. Peptide mapping and amino acid sequencing were used in the identification. This desamido form (hGHAsp152) could be produced by incubation of the undeamidated hormone in an alkaline medium. Another minor deamidated form which contained glutamic acid at 137 (hGHGlu137) also was identified in preparations of hGH. This form was not produced by alkaline treatment of hGH. Limited hydrolysis of hGH with subtilisin produced two cleaved forms, one with cleavages at positions 139 and 149 and another with cleavages at 139 and 146. hGHAsp152 underwent only one type of modification, cleavage at positions 139 and 146. Hydrolysis of hGHGlu137 resulted in cleavages in the region of 139 to 149 identical with those noted with hGH, but in addition, proteolysis had occurred in the region of 95 to 127, an area where hGH was not attacked by subtilisin. That Glu at 137 modified cleavage points was also indicated by the greater resistance of hGHGlu137 to hydrolysis by subtilisin as compared to hGH. The results demonstrate that deamidation can alter points of proteolytic cleavage. If proteolytic processing of hGH is found to be of physiologic significance, deamidation may be a way of directing specific cleavages.

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 CaCl 2 at 37°C decreased the fluidity, as indicated by positive increases in the order parameters. The ordering effect of Ca 2+ 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 45Ca 2+, and our results suggest that Ca 2+-induced alterations in the lipid fluidity involve cation binding to low-affinity sites. The cellular movements of Ca 2+ and, in particular, the binding of Ca 2+ to the plasma membrane may play important roles in insulin's 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 Ca 2+. The fluidities of I(12,3)-labeled rat adipocyte ghosts or human erythrocyte ghosts were also unaffected by various forms of human growth hormone.

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. In vitro experiments utilizing epididymal adipose tissue from hypophysectomized rats indicated that 20K was unable to accelerate glucose utilization as measured by glucose uptake and CO 2 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.

Modified forms of human growth hormone with increased biological activities.

A proteolytically modified form of human growth hormone, isolated from a pituitary extract, had a growth promoting potency of 6.9 IU/mg in the tibial line assay. The prolactin activity of the material, measured by the crop sac assay, was 14.5 IU/mg. Peptide mapping indicated that residues 135–146 were missing from the large disulfide loop of the protein. Data are presented also for two other modified forms of the hormone. One lacked residues 135–140; the other was missing residues 135–145 together with a number of amino acids COOH-terminal to the cleavages. These latter two forms also showed a marked increase in prolactin activity and some potentiation of growth stimulating ability. All three forms were indistinguishable from intact growth hormone by radioimmunoassay and did not react with antiserum to human prolactin. These results indicate that the most active form of human growth hormone, as measured by the tibial line and crop sac assays, is not the intact hormone. (Endocrinology 94: 883, 1974)