Biomedical subjects

A Gertler

Publications and source records attributed to A Gertler.

At least 91 records · Page 5Linked to original sources

PubMed1992

Preparation, purification, and determination of the biological activities of 12 N terminus-truncated recombinant analogues of bovine placental lactogen.

Removal of 13 to 15 amino acids from the N terminus of bovine placental lactogen (bPL), which according to the three-dimensional structure of pGH corresponds to a nonhelical part of bPL, did not effect its secondary structure or change the monomer content of the protein preparation. However, it remarkably decreased the binding of the prolactin (PRL) type of receptors on Nb2 cells with subsequent reduction in bioactivity. The binding to the growth hormone (somatogen) receptors either did not change or was increased, resulting in an increase of somatogen receptor-mediated bioactivity. Further truncation (17-18 amino acids) resulted in a decrease of alpha-helical content and loss of binding properties and biological activity mediated through interaction of the analogues with both somatogen (3T3-F442A cells) and lactogen (PRL) receptors (NB2-11C cells). Truncation of 19-27 amino acids caused additional loss in activity, without further change in the secondary structure. Replacement of Leu28 by a more hydrophobic Phe has only minor, if any, effect on the bioactivity of bPL. Occasional point mutations due to polymerase chain reaction errors in several analogues did not seem to have any major effect on the hormone properties. It can thus be suggested that the N-terminal part of the nonhelical portion of bPL, which corresponds to the portion of the molecule that does not exist in growth hormones, is required for efficient binding to the lactogen (PRL) but not to the somatogen or unique bPL receptors. Removal of the N-terminal part of pBL changed the specificity of bPL by decreasing its PRL receptor-mediated activities and increasing its somatogen receptor-mediated activities.

Amino Acid Sequence↗

PubMed1992

Oral absorption of biologically active insulin in common carp (Cyprinus carpio L.).

1. Bovine insulin dissolved in 0.05 M deoxycholic acid was absorbed through oral intubation in fish weighing 200-300 g. 2. The peak of absorption appeared in all fish, 30-45 min after intubation and was followed by a gradual decrease. The extent of absorption was extremely variable, with up to a 20-fold difference between individuals. No detectable insulin was found in fish intubated with vehicle. 3. The absorbed hormone retained 17-88% of its lipogenic bioactivity in vitro. The absorbed insulin lowered the levels of several amino acids in the intubated fish, indirectly indicating that its bioactivity was also retained in vivo.

Absorption↗

PubMed1992

Characterization of insulin-like growth factor binding proteins secreted by cultured bovine theca and granulosa cells.

Insulin-like growth factor binding proteins (IGFBPs) secreted by bovine granulosa and theca interna cells cultured in the presence of different luteinizing factors--insulin (2 micrograms/ml), forskolin (10 microM), or a combination of the two were examined and characterized. Direct binding of [125I]IGF to the conditioned media was compared to progesterone production under these different treatments. In theca cells, maximal secretion of IGFBPs was achieved using forskolin alone, whereas maximal progesterone production was induced by the insulin+forskolin treatment. In contrast, maximal secretion of both IGFBPs and progesterone in granulosa cells was achieved using forskolin alone. IGFBP species secreted by the two cell types under the different treatments were detected by ligand blotting. Conditioned media from theca cells in serum-free medium collected on the seventh day of culture exhibited three bands of 34, 40 and 44 kDa when treated with insulin or forskolin. The intensity of the 40-44 kDa complex was enhanced and a 21 kDa band appeared when cells were treated with a combination of insulin plus forskolin. Conditioned media of granulosa cells stimulated with insulin or forskolin exhibited 21, 27, 29, 34 and 40-44 kDa bands. Treatment with insulin+forskolin greatly increased the intensity of a 40-44 kDa complex. A similar shift towards high molecular weight binding proteins was observed when these media were analyzed by high-performance liquid chromatography gel filtration. These findings substantiate the secretion of IGFBPs by bovine theca and granulosa cells and show it to be dependent on culture treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Comparative mitogenic and galactopoietic effects of IGF-I, IGF-II and Des-3-IGF-I in bovine mammary gland in vitro.

Insulin-like growth factors (IGFs) I and II (IGF-I, IGF-II) and Des-3-IGF-I at physiological concentrations are potent mitogens of bovine undifferentiated mammary epithelial cells cultured in collagen in a serum-free medium. Des-3-IGF-I was found to be as potent as IGF-I, while IGF-II was significantly less active. All three factors acted either synergistically or additively with epidermal growth factor (EGF), cholera toxin and fetal calf serum (FCS). Indirect evidence indicates that despite its lower mitogenic activity the action of IGF-II is mediated through IGF-I receptors. The galactopoietic activity of Des-3-IGF-I and IGF-II was studied in an organ culture of bovine lactating mammary glands using lactogen-responsive fat synthesis as a test. Neither Des-3-IGF-I nor IGF-II exhibited galactopoietic activity nor did they affect the galactopoietic activity of prolactin.

Animals↗

PubMed1992

Evidence that the N-terminus of human growth hormone is involved in expression of its growth promoting, diabetogenic, and insulin-like activities.

Recent work with various point and deletion mutants of human GH (hGH) has suggested that the proximal N-terminal end of the hormone molecule is important for its growth promoting action. This study was conducted to examine the growth promoting, diabetogenic, and insulin-like activities of two N-terminal mutants of hGH, the deletion mutant Des-7 hGH (met8, ala11), and a chimeric mutant of bovine GH (bGH) and hGH containing the N-terminal 13 amino acids of bGH (met, ala 1-13/14-191, asp11). The CD spectra of these mutants are similar to that of wild-type hGH and they retain lactogenic activity on Nb2 lymphoma cells, whereas their ability to bind to somatogenic receptors on IM-9 lymphocytes and bovine liver membranes is markedly reduced. In this study, growth promoting activity of the mutants was assessed using the 9-day weight gain test in hypophysectomized rats. Des-7 hGH had a potency of 0.03 IU/mg protein in this assay, whereas the potency of the bGH/hGH chimera was 0.71 IU/mg. Diabetogenic activity was tested in the ob/ob mouse, using the elevation of fasting blood glucose and the worsening of glucose tolerance after a 3-day course of treatment as end-points. Both Des-7 hGH and the bGH/hGH chimera had reduced diabetogenic activity compared to that of biosynthetic wild-type hGH, consistent with their reduced growth activity. Insulin-like activity was assessed by testing the in vitro ability of the mutants to stimulate [14C] glucose oxidation by epididymal adipose tissue of hypophysectomized rats. Des-7 hGH had about 1% the activity of wild-type hGH, whereas the chimera was about 20% as active. When Des-7 hGH was added to the incubation medium along with wild-type hGH in ratios of 5, 12.5, or 25:1 (Des-7 hGH:hGH), the insulin-like action of hGH was significantly inhibited, indicating that the mutant is a modest antagonist of the insulin-like action of hGH. When the ability of Des-7 hGH to compete with [125I] hGH for binding to isolated rat adipocytes was tested, the mutant was about 10% as effective as wild-type hGH. Thus, Des-7 hGH appears to be more effective in binding to adipocyte GH receptors than in triggering an insulin-like response, perhaps accounting for its modest antagonistic activity. The results of this study suggest that the proximal N-terminal end of the hGH molecule is involved in the expression of the growth promoting, diabetogenic and insulin-like activities of GH.

Adipose Tissue↗

PubMed1992

Short-term in vivo regulation of prolactin receptors in the liver, testes, kidneys, and mammary gland of rats.

Infusion of 17-beta-estradiol for 3 h caused a more than 100% increase in the number of PRL receptors (PRL) in the microsomal liver fraction of male rats without affecting the hormone-receptor affinity. A small but significant increase in PRL receptors also was found in testes but not kidneys. Testosterone infusion to female virgin rats for a similar time period resulted in some nonsignificant decreases in PRL receptors in the liver but not the mammary gland or kidneys. Testosterone infusion to males or 17-beta-estradiol infusion to females did not change the number or affinity of PRL and growth hormone receptors. The level of serum PRL was not influenced during infusion with both hormones.

Animals↗

PubMed1991

Biological activity of a fluorescein human growth hormone derivative prepared by specific covalent labeling of lysine-70.

Modification of human growth hormone (hGH) with a low equimolar concentration of fluorescein isothiocyanate (FITC) yielded a derivative containing 1 mol of fluorescein/mol of protein. The site of modification was identified as lysine-70. Lysine-70 of hGH is about 3-fold more reactive than a "normal" lysine in a protein, having pseudo-first-order kinetics Kobs = 110 +/- 7 M-1 min-1 at pH 10.5. The pKa of the lysine was estimated to be 10.7, within the normal range of normal epsilon-lysine moieties in proteins. This higher chemical reactivity seems to favor selective labeling of this moiety at low FITC concentrations. To obtain monomodified derivatives, hGH was derivatized with 0.6 equiv of FITC, and the modified derivatives were separated from unreacted hormone by means of HPLC using a Mono Q column. Its biological activity, determined by Nb2 bioassay, decreased to 40%, and its affinity toward lactogen receptors in Nb2 cells and toward somatogen receptors in bovine liver decreased respectively to 30% and 20%. The present study indicates that out of the seven amino groups of human growth hormone, the epsilon-amino group of lysine-70 is excessively reactive toward FITC. Second, this particular amino group contributes to receptor binding and receptor activation. Lysine-70 is located in the loop between the first and second helix and close to the carboxy-terminal end of the first helix. This contribution is most likely the result of the formation of an electrostatic interaction between the hormone and the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗

PubMed1991

Absorption of bioactive human growth hormone after oral administration in the common carp (Cyprinus carpio) and its enhancement by deoxycholate.

Recombinant human growth hormone was administered orally to carp and serum levels of absorbed bioactive hormone were investigated using a highly sensitive Nb2 rat lymphoma cell bioassay and radioimmunoassay. Serum levels of bioactive hGH reached maximum values 30 min after oral intubation and then gradually decreased. Co-administration of the hormone with deoxycholate to fasted carp resulted in up to a 1000-fold increase in absorption compared to aqueous solutions of the hormone, but had no effect on the kinetics of the absorption process. Absorption of the hormone in starved fish was significantly greater than in fed fish. A linear dose-response relationship was observed for hGH in starved fish and the level of absorption in fed fish was influenced by the time interval from the last meal. The ratio of bioactive to immunoactive hGH in fasted fish indicated little loss of bioactivity and also that deoxycholate may be protective against hGH degradation. The present study demonstrates for the first time that biologically active hGH is absorbed in the common carp after oral intubation. Furthermore, the use of a biological detergent dramatically increased the extent of hGH absorption. Additional studies are required to establish the appropriate conditions (diet composition, feeding level, and frequency, etc.) in which polypeptide hormones could be introduced orally to fish.

Administration, Oral↗

PubMed1991

Distinct placental lactogen and prolactin (lactogen) receptors in bovine endometrium.

Specific binding sites for bovine placental lactogen (bPL) and the lactogenic hormone, prolactin, have been detected in endometrial membranes isolated from uteri of mid-pregnant heifers. The specific binding of human growth hormone (hGH) (used to monitor the presence of lactogenic binding sites) and of bPL was increased approximately 4-fold following treatment of the membranes with 4 M MgCl2. Binding was found to be ligand specific, membrane protein concentration-, time- and temperature-dependent and reversible. Scatchard analysis of bPL and hGH competition binding data revealed curvilinear plots with dissociation constants for the high affinity sites of 4.1 x 10(-11) M and 6.4 x 10(-11) M, respectively. The maximum capacity of binding of bPL at the high affinity site was 21 fmol/mg). membrane protein while approximately twice the level of binding was measured for hGH (39 fmol/mg). Both hGH and bGH, but not ovine prolactin, competed with [125I]bPL for binding. The concentrations of hGH and bGH needed to effectively compete were however 100-fold higher than those required for unlabeled bPL. No specific binding of radiolabeled bGH was detected in endometrial tissue suggesting the absence of bGH receptors. Preferential competition of [125I]hGH binding was observed by prolactin and bPL. From these data it may be inferred that hGH binding is indicative of the presence of both lactogenic (prolactin) and bPL binding sites in endometrial tissue. The presence of distinct bPL receptors in the endometrium from mid-pregnant cows suggests a possible role for bPL in the maintenance of pregnancy.

Animals↗

PubMed1991

Hydroxyphenyl acetate derivatives inhibit protein tyrosine kinase activity and proliferation in Nb2 rat lymphoma cells and insulin-induced lipogenesis in rat adipocytes.

The ortho, meta, and para forms of hydroxyphenyl acetate were found to be inhibitory in the order of ortho greater than para greater than meta in three distinct biological assays: (a) insulin-dependent assimilation of glucose into lipids in intact adipocytes, (b) growth and proliferation of Nb2 rat lymphoma cells, and (c) tyrosine phosphorylation of copolymer (Glu4Tyr) under cell-free conditions. Although relatively high concentrations of o-hydroxyphenyl acetate (OHPA) were required to inhibit these processes, the inhibitor exhibited a low index of cytotoxicity and high specificity toward inhibiting tyrosine- (but not serine-) specific kinases. Cell cycle analysis of the DNA histograms in Nb2 cells revealed that exposure to OHPA did not change the initiation of the G0/G1----S transition but drastically reduced its rate and a subsequent cell proliferation. Kinetic experiments in which the inhibitor was added or withdrawn through different phases of cell cycle confirmed this conclusion. OHPA inhibition of cell growth appears to be limited to eukaryotic cells as the growth of either gram-positive or gram-negative bacteria was unaffected by the presence of the inhibitor. The study supports the following conclusions: (a) Events that are dependent on tyrosine phosphorylation are indeed essential for mammalian cell growth and proliferation. (b) Neither the initial nor intermediate events of the proliferative cascade that occur in the Nb2 cells prior to DNA synthesis are dependent on the activity of protein tyrosine kinase(s) that are inhibited by OHPA. (c) Cell growth of prokaryotic cells and yeast may lack protein tyrosine kinase activity or be less dependent on events requiring tyrosine phosphorylation. (d) Inhibition of the insulin-dependent lipogenesis is subsequent to the inhibition of insulin receptor tyrosine kinase activity.

Adipose Tissue↗

PubMed1991

Bovine pituitary, kidney, uterine and mammary gland extracts contain bovine mammary epithelium growth factors that synergise with IGF-I and fetal calf serum: indication for involvement of GTP-binding proteins.

Bovine mammary undifferentiated epithelial cells from young female calves, cultured in three-dimensional collagen gels in serum-free medium exhibited ultrastructural organization that resembled the in vivo situation. Extracts of bovine pituitary, kidney, uterus and mammary gland, stimulated cell proliferation in a dose-dependent manner. This mitogenic activity strongly synergised with the existant growth factors (GFs) in FCS and with IGF-I, while the addition of EGF had only minor effect. No synergistic manifestation was found with cholera toxin but pertussis toxin inhibited the growth-promoting activity of all four extracts. Other experiments indicated that this mitogenic activity does not result from prolactin, growth hormone or fibroblast growth factor. The present and former results, in which synergism between IGF-I and cholera toxin was demonstrated, suggest therefore, that the mitogenesis of normal mammary epithelial cells regulated by several tissue derived growth factors, consists of at least two pathways which are distinct from those activated by EGF and IGF-I. One of these pathways indicates involvement of pertussis toxin-sensitive GTP-binding proteins, and the other, activation of cholera toxin-sensitive adenylate cyclase.

Animals↗

PubMed1991

Serum levels and peritoneal loss of prolactin in CAPD patients.

We studied the serum levels and peritoneal transport of prolactin in 13 CAPD patients and compared with 8 patients on hemodialysis and 10 normal subjects matched by age (30-78 years), sex, body mass index and serum glucose concentrations. CAPD and hemodialysis patients were matched also by hematocrit, serum creatinine, albumin concentrations and duration and dialysis (0-79 months), the prolactin levels in serum and in effluent were measured by RIA and NB2 bioassay. CAPD patients had higher serum prolactin levels than did hemodialysis-treated patients and control subjects. The bioactive/immunoactive ratio of serum prolactin in CAPD patients was 0.7 +/- 0.1 versus 0.9 +/- 0.1 in the control subjects (P less than 0.05) and 0.8 +/- 0.04 in the hemodialysis patients, mean +/- SEM. A significant linear correlation was demonstrated between serum levels and 8 hr peritoneal mass transfer of this hormone. A notable drop of prolactin took place after the first hour of dialysis. Serum prolactin was not influenced by peritoneal protein loss, glucose absorption rate or duration of CAPD. From this study we may conclude that hyperprolactinemia associated with CAPD is not affected by continuous peritoneal loss of prolactin, demonstrating only a slight decrease in bioactivity.

Adult↗

PubMed1990

Mitogenic and binding properties of monoclonal antibodies to the prolactin receptor in Nb2 rat lymphoma cells. Selective enhancement by anti-mouse IgG.

Three monoclonal antibodies (mAbs) (T6, U5, and U6) against prolactin (PRL) receptors in rat liver were studied in the rat lymphoma lactogen-dependent (Nb2-11C) and autonomous (Nb2-SP) cell lines. The mAbs had strong affinity for lactogen receptors (Ka = 12-14 nM-1), similar to that of human growth hormone (hGH) which is a lactogenic hormone. T6 and hGH competed for the same binding site, while U5 and U6 interacted with another epitope. The 125I-hGH-receptor complex could be immunoprecipitated by either U5 or U6, but not by T6. Affinity labeling and immunoblotting revealed that hGH and U6 bind to a protein of 63-65 kDa. T6, U5, and U6 were mitogenic in Nb2-11C cells but their respective potencies were 185-, 70-, and 4700-fold lower than that of hGH. Anti-mouse IgG enhanced the mitogenic effect of all three mAbs and almost completely abolished the differences between them, although their mitogenic activity was still 60-120-fold lower than hGH. Des-13-hGH, a competitive antagonist of hGH which hardly effected the binding of 125I-U5, inhibited the U5-stimulated proliferation of Nb2-11C cells in a noncompetitive manner, indicating that simultaneous binding of both ligands fixed the receptor in a nonactive conformation. A Fab fragment of T6 was not mitogenic, and inhibited the hGH-induced mitogenesis in a competitive manner, but its mitogenicity could be restored by anti-mouse IgG. We suggest that the dimerization or oligomerization of the lactogen receptor in Nb2-11C cells is an obligatory step in the transduction of the mitogenic signal. It may be induced by binding of the mAb to a site, which can be either identical or may even be distinct from that which binds the lactogenic hormone.

Animals↗

PubMed1990

Proliferation of bovine undifferentiated mammary epithelial cells in vitro is modulated by G-proteins.

Several cAMP-elevating agents such as cholera toxin (CT), forskolin and 3-isobutyl-1-methylxanthine (IBMX) exhibited weak mitogenic activity on bovine undifferentiated mammary epithelial cells in three-dimensional collagen culture. CT and IBMX strongly synergized with epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) or both, but not with 10% fetal calf serum (FCS). Permeable cAMP analogs also synergized with IGF-I. Other hormones such as ovine prolactin, bovine growth hormone, estrogen or progesterone were not mitogenic and not synergistic with EGF, IGF-I, CT and FCS. Pertussis toxin (PT) reduced the DNA synthesis in cells cultured in the basal medium and attenuated 40-90% of the mitogenic activity stimulated by 10% FCS. PT inhibition of DNA synthesis was accompanied by ADP-ribosylation of 40 kDa and 41 kDa membrane proteins. The 41 kDa protein cross-reacted with antibodies that recognize the Gi-protein of the adenylate cyclase system, indicating the involvement of the latter in the mitogenic process. The nature of the second protein remains unknown. The present results suggest that the mitogenesis of normal mammary epithelial cells which is stimulated by IGF-I, EGF and other factors found in FCS is mediated through both cAMP-dependent and independent pathways. These pathways include PT-sensitive GTP-binding proteins.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Site-directed mutations of human growth hormone that selectively modify its lactogenic activity and binding properties.

Two novel analogs of human (h) GH, 1) Des-7-hGH (Arg8Met, Asp11Ala) in which the Arg8 was substituted by Met and Asp11 by Ala, and 2) bovine (b) GH/hGH hybrid II (MetAla 1-13/14-191, Ala11Asp) composed of 13 N-terminal amino acid of bGH and elongated by two amino acids (Met-Ala-1-13) and 14-191 amino acids of hGH, were constructed and expressed in Escherichia coli. CD spectra indicated that the alpha-helix content of the purified proteins was similar to that of the native hormone. Both analogs retained their full ability to stimulate the proliferation of Nb2 lymphoma cells, and their binding to the lactogen receptors in homogenate of Nb2 cells and in microsomal fraction from bovine lactating mammary gland was only slightly reduced. However, their ability to bind to the somatogen receptors in intact IM-9 lymphocytes and bovine liver was reduced by 7- to 11-fold (bGH/hGH hybrid II) and 20- to 30-fold (Des-7-hGH). Both analogs were able to down-regulate the respective lactogen and somatogen receptors in intact Nb2 and IM-9 cells. The galactopoietic activity of both analogs in the lactating bovine mammary explants bioassay was almost completely abolished, and the bGH/hGH hybrid II exhibited a remarkable antagonistic activity. These results further indicate that the lactogen receptors in different species or organs are not identical. We have shown that the new recombinant analogs of hGH that recognize both somatogen and lactogen receptors but have modified postreceptor effects are helpful in elucidating these differences.

Base Sequence↗

PubMed1989

Solubilization and characterization of lactogenic hormone receptor from kidney of lactating cow.

Solubilization of the microsomal fraction from bovine kidney by Triton X-100 or by 3-[(3-cholamidopropyl)-dimethylammonio] 1-propanesulfonate (CHAPS) increased 2-fold the thermodynamic association constant for hGH. While solubilization with CHAPS did not change the 13-fold preferential binding of human growth hormone (hGH) over ovine prolactin (oPRL), solubilization with Triton X-100 increased this preference to 47-fold. The binding was optimal at pH 7-7.5 in the presence of 10 mM of MgCl2 or CaCl2. The association rate with hGH was identical in the microsomal and Triton X-100 solubilized fractions but the dissociation was slower in the latter. Only partial dissociation was observed at neutral pH. Full dissociation was, however, achieved by lowering the pH to 4-5, indicating that the binding was not covalent. Gel filtration studies of the Triton X-100 solubilized fraction after preincubation in the presence of reducing agent revealed two sharp peaks of activity, one having Mr of greater than 700 kDa that represented the aggregated receptor, and the second, with Mr 110-115 kDa. The specificity of the partially purified receptors clearly shows that they are lactogenic and not somatogenic. They resemble lactogenic receptors found in other bovine organs, but differ from other species particularly in their differential affinities of PRL and hGH.

Animals↗

PubMed1989

Indication of different lactogen and somatogen binding sites in the human growth hormone molecule as probed with monoclonal antibodies.

The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interaction has been investigated using as probes monoclonal antibodies (Mabs) to hGH of defined epitope specificity profile. Seven high affinity Mabs were studied for their ability (i) to inhibit the binding of 125I-hGH to Nb2-SP rat lymphoma cells and to IM-9 human lymphocytes possessing lactogen and somatogen type receptors, respectively; and (ii) to interfere with the hormone (hGH or Met8Leu hGH)-induced proliferation in Nb2-11C lymphoma cells. The ability of these Mabs to inhibit the 125I-hGH binding and the hormone-induced proliferation in Nb2-11C cells was negatively correlated with the ability of these Mabs to cross-react with met14 hGH. Furthermore, Mabs Nos. 3 and 7, which cross-reacted minimally (0.2-0.4%) with Met8Leu hGH, were unable to interfere with the mitogenic activity of Met8Leu hGH in Nb2-11C cells. These results indicate that the first 13 amino acids of the N-terminal region of hGH are necessary for its lactogen activity. The inhibition of 125I-hGH binding to IM-9 cells by these Mabs was similar to those observed in Nb2-SP cells, except for Mabs Nos. 19 and 1. These Mabs inhibited more strongly the binding of 125I-hGH to IM-9 than to Nb2 cells and recognized antigenic epitopes close to the C-terminal part of the molecule. These results suggest that the somatogen receptor binding site of hGH may be located on two sites, one at the N-terminal and the other one close to the C-terminal, while the lactogen receptor is mainly confined to the N-terminal part.

Animals↗

PubMed1989

Chimeric bovine-human growth hormone prepared by recombinant DNA technology: binding properties and biological activity.

A chimeric bovine GH (amino acids Met-Asp-Gln-greater than 1-23) and human GH (hGH) (amino acids 24-191) plasmid was constructed and expressed in Escherichia coli. The purified protein (chimeric GH) exhibited a 2-3 order of magnitude lower affinity toward lactogenic receptors in Nb2 lymphoma cells, microsomal fractions from bovine mammary gland and male rat liver. The affinity towards somatogenic receptors in IM-9 human lymphocytes and male rat liver was decreased to a much lesser degree. This diminished affinity towards lactogenic receptors was accompanied by a parallel decrease in the ability of the chimeric GH to stimulate proliferation of Nb2-11C lymphoma cells and the lipogenesis in bovine mammary gland. This implies that occupation of the respective receptors by either chimeric GH or hGH leads to identical postreceptoral effects. The chimeric GH was also capable of down-regulating the lactogenic receptors in Nb2 lymphoma cells and was recognized by three anti-hGH monoclonal antibodies. These and previously published results indicate that the N-terminal part of hGH is essential for the high affinity binding to lactogenic receptors and subsequent biological effect. Removal or replacement by a corresponding part of bovine GH converts the hormone, respectively to weak antagonist or agonists. Analysis of our data, based on hydropathy index leads us to suggest that the high affinity binding site of the hGH towards lactogenic receptors is mainly confined to amino acids nos. 8-18.

Animals↗