Nanoclusters of crystallographically aligned nanoparticles for magnetic thermotherapy: aqueous ferrofluid, agarose phantoms and ex vivo melanoma tumour assessment.

Magnetic hyperthermia is an oncological therapy where magnetic nanostructures, under a radiofrequency field, act as heat transducers increasing tumour temperature and killing cancerous cells. Nanostructure heating efficiency depends both on the field conditions and on the nanostructure properties and mobility inside the tumour. Such nanostructures are often incorrectly bench-marketed in the colloidal state and using field settings far off from the recommended therapeutic values. Here, we prepared nanoclusters composed of iron oxide magnetite nanoparticles crystallographically aligned and their specific absorption rate (SAR) values were calorimetrically determined in physiological fluids, agarose-gel-phantoms and ex vivo tumours extracted from mice challenged with B16-F0 melanoma cells. A portable, multipurpose applicator using medical field settings; 100 kHz and 9.3 kA m-1, was developed and the results were fully analysed in terms of nanoclusters' structural and magnetic properties. A careful evaluation of the nanoclusters' heating capacity in the three milieus clearly indicates that the SAR values of fluid suspensions or agarose-gel-phantoms are not adequate to predict the real tissue temperature increase or the dosage needed to heat a tumour. Our results show that besides nanostructure mobility, perfusion and local thermoregulation, the nanostructure distribution inside the tumour plays a key role in effective heating. A suppression of the magnetic material effective heating efficiency appears in tumour tissue. In fact, dosage had to be increased considerably, from the SAR values predicted from fluid or agarose, to achieve the desired temperature increase. These results represent an important contribution towards the design of more efficient nanostructures and towards the clinical translation of hyperthermia.

The granulocyte colony-stimulating factor (G-CSF) upregulates metalloproteinase-2 and VEGF through PI3K/Akt and Erk1/2 activation in human trophoblast Swan 71 cells.

INTRODUCTION: Although the expression of the granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) in placental tissues suggests that the cytokine could play a role in placental development, the relevance of G-CSF:G-CSFR interaction in trophoblast cells remains to be studied. Thus, the possible functional role of G-CSF was examined in a human trophoblast cell line (Swan 71 cells). METHODS AND RESULTS: The expression of G-CSFR was detected by immunocytochemistry and Western blot assays. G-CSF treatment exerted neither a proliferative nor a protective effect on H2O2-mediated cell death in trophoblast cells. Gelatin zymography of supernatants collected from G-CSF-treated cells showed an increment of metalloproteinase-2 (MMP-2) activity. We also found higher MMP-2 and VEGF expression levels in conditioned medium from cells exposed to G-CSF. In addition, it was demonstrated that G-CSF induced the activation of PI3K/Akt and Erk1/2 pathways, which in turn activated NF-kB. By using selective pharmacological inhibitors, it was showed that these pathways are mediating the biological effects produced by G-CSF in Swan 71 cells. DISCUSSION AND CONCLUSION: We have demonstrated for the first time that G-CSF increases MMP-2 activity and VEGF secretion in Swan 71 cells through activation of PI3K/Akt and Erk signaling pathways, both contributing to the translocation of NF-kB to the nucleus. These data suggest that G-CSF is involved in the regulation of trophoblast function, and should be considered as a locally produced cytokine probably contributing to embryo implantation and the development of a functional placenta.

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells.

Properties of cryptic epitopes and their corresponding antibodies as indicated by the study of human and ovine growth hormones.

Antibodies (Ab) directed to hidden antigenic determinants (cryptotopes) are undesirable because they are not neutralizing. Additionally, we have previously demonstrated a close association between the extent of Ab to cryptic determinants and the expression of autoantibodies (autoAb) under some experimental conditions. Thus, the first objective of this work was to establish the physicochemical characteristics of Ab to cryptotopes and the second one was to examine the structural features of cryptic epitopes themselves. Using human and ovine growth hormones (hGH and oGH) as antigenic models and competition ELISA under different conditions of temperature, pH or ionic strength, we did not find any difference between the binding properties of anti-cryptic epitope antibodies (Ab) and anti-native epitope Ab. Then, using synthetic peptides and tryptic digests and direct and competition ELISAs we studied the structures of cryptic hGH and oGH epitopes. Isolated peptides either in solution or adsorbed on microplates failed to react. Partially digested hGH was recognized only when insolubilized on microplates, and anti-oGH Ab only reacted with a large fragment of the hormone either in solution or insolubilized. These results indicate that, at least in the case of hGH and oGH, cryptic epitopes are not simple linear sequences, as commonly referred without any evidence, but new exposed conformational structures different from those found in the native antigen.

Monoclonal antibodies inducing conformational changes on the antigen molecule.

Monoclonal antibodies (MoAbs) are extensively used as biological tools because of their invariable specificity. However, the interpretation of results can be misled by the behaviour of MoAb displaying allosteric effects, i.e. long-range conformational changes on the antigen (Ag). It has been shown that some MoAbs are able to modify the spatial structure of the corresponding protein Ag, affecting in this way its biological activity as well as its binding to a second MoAb. Thus, a researcher using a MoAb as a tool to investigate some features of an antigenic molecule must be aware of the possible positive or negative allosteric properties of the antibody.

Effects of various adjuvants and a viral infection on the antibody specificity toward native or cryptic epitopes of a protein antigen.

An immunization protocol that induces antibodies (Abs) directed to cryptic epitopes of a protein antigen (Ag) reduces the efficacy of vaccines that ideally should induce Abs against native epitopes. We have shown earlier that viral infections concomitant with immunization against a protein tend to shift the Ab specificity toward cryptic epitopes and tend to induce the production of autoantibodies (autoAbs). Here, we show the effects of three adjuvants on the Ab specificity in the absence or presence of a viral infection (lactate dehydrogenase-elevating virus or LDV), with human growth hormone (hGH) being, as before, the protein Ag. Pathogen-free CBA/Ht and BALB/c mice were immunized with hGH in the presence of complete Freund's adjuvant (CFA), monophosphoryl lipid A (MPL) or alum, with the animals being either infected with LDV or not infected with LDV. Conventional and competition enzyme-linked immunosorbent assays (ELISAs) indicated that in noninfected mice, CFA induced higher titres of anti-hGH Ab than did MPL or alum, with the Ab being almost totally directed to cryptic hGH epitopes. Strikingly, CFA plus LDV infection in CBA/Ht mice shifted the specificity of the anti-hGH Ab toward native epitopes, whereas the virus decreased the Ab titre when MPL or alum was used. Our Western blot results showed that 70% of mice immunized with hGH in the presence of any adjuvant produced autoAbs against a variety of tissue Ags. The amount of autoAb and the concentration of Ab to hGH cryptic epitopes did correlate, suggesting a relationship between both kinds of Ab. Significant differences were observed in the various effects of adjuvants and the viral infection between the two mouse strains used in this work.

Relative localization of the prolactin receptor binding sites for lactogenic hormones.

A monoclonal antibody termed MAb R7B4, directed to an epitope present in prolactin receptors (PRLRs), was used as a tool to map the receptor binding sites for human growth hormone (hGH), ovine prolactin (oPRL) and human placental lactogen (hPL). Although the three hormones completely inhibited the binding of each other to Nb2 cells or rat liver receptors, MAb R7B4 behaviour was different depending on the hormone tested and the receptor source. According to the MAb effects, PRLR from Nb2 cells would locate both hGH and oPRL close to R7B4 epitope, whereas hPL would bind far from the MAb binding site. On the other hand, PRLR from rat liver should bind hGH close to the R7B4 epitope but oPRL and hPL would be recognized by a separate region of the same receptor. Thus, results presented in this paper suggest that PRLR binding sites for hGH, oPRL and hPL do not exactly overlap in spite of full competition between ligands.

Conformational and sequential epitopes on the human granulocyte-colony stimulating factor molecule (hG-CSF) and their role in binding to human placenta receptors.

Monoclonal antibodies (mAbs) named 8C2 and 6E3, directed against the recombinant human granulocyte colony-stimulating factor (hG-CSF), were used as probes to study the cytokine orientation on its binding to receptors from human placenta. Competition enzyme linked immunoabsorbent assays (ELISA) revealed that mAb 8C2 would be directed to a linear epitope, whereas mAb 6E3 would delimit a more assembled epitope. Gel-filtration high performance liquid chromatography (HPLC) of the immune complexes formed by incubating [(125)I]hG-CSF with each mAb showed that epitope 8C2, but not 6E3, was altered after cytokine iodination. In addition, mAb 6E3 completely inhibited [(125)I]hG-CSF binding to human placental microsomes. Although [(125)I]mAb 6E3 was unable to bind to preformed hG-CSF-receptor complexes, [(125)I]mAb 8C2 did recognize hG-CSF previously bound to receptors, suggesting that epitope 8C2 would remain accessible in the hG-CSF-receptor complex. To identify the cytokine region defined by mAbs, hG-CSF was digested with different proteolytic enzymes: Arg-C, Glu-C, trypsin and alpha chymotrypsin. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation. Results showed that mAb 6E3 would be directed to a conformation-dependent epitope located close to the hG-CSF binding domain and included into the sequence 1-122/123, whereas mAb 8C2 recognized the region 41-58, which represents a linear epitope left exposed after cytokine binding to receptors from human placenta.

Suitable experimental conditions are required to characterize interferon-alpha2b synthetic peptides.

The binding and antiproliferative activities of synthetic peptides 29-35 and 122-139 of interferon-alpha2b, both of which contain a cysteine residue in their sequences, were studied in the presence or absence of a dissociation medium containing mainly urea, dithiothreitol and 2-mercaptoethanol. Although interferon-alpha2b peptides either did not modify or slightly increased 125I-labelled interferon-alpha2b specific binding to WISH cell-membrane receptors in the absence of dissociation medium, significant binding inhibition was obtained when both peptides were assayed in dissociation medium. Furthermore, also in the presence of dissociating agents, the two fragments inhibited cell growth in a concentration-dependent manner, the 122-139 sequence being more effective than the 29-35 sequence. No additive effect on interferon binding and cell proliferation was observed when both peptides were added simultaneously. Results obtained after submitting peptide 122-139 to gel filtration or PAGE under different experimental conditions showed the presence of dimers and/or noncovalent aggregates arising from intermolecular disulfide bridges or hydrophobic interactions. Thus, our results indicated that peptide effects on 125I-labelled interferon-alpha2b binding and WISH cell proliferation were clearly manifested when the amount of monomeric species increased, showing that suitable experimental conditions should be used to study peptide behavior. The ability of both peptides to effectively trigger an interferon-specific biological action, such as cell growth inhibition, strongly suggested that 29-35 and 122-139 interferon-alpha2b fragments constitute the conformational epitope or mimotope that interacts with the cytokine-specific receptor.

Positive cooperative effects between receptors induced by an anti-human growth hormone allosteric monoclonal antibody.

Monoclonal antibodies (MAb) anti-human growth hormone (hGH) termed MAb AE5, AC8 and F11 recognize a cluster of epitopes left exposed after hormone binding to receptors. Since these MAb were able to produce either positive (MAb AE5) or negative (MAb AC8 and F11) allosteric effects on hGH binding, the purpose of this work was to further characterize MAb behavior. Results indicated a straight correlation between MAb allosteric effects and affinity constant values for binding of different hGH:MAb complexes to lactogenic receptors from rat liver. Affinity of hGH:MAb AE5 as well as hGH:Fab AE5 complexes enhanced proportionally to the fraction of occupied receptors and Hill coefficients higher than 1 were obtained, suggesting the induction of positive cooperative effects between membrane-bound receptors. On the other hand, hGH:MAb AC8 and hGH:MAb F11 complexes binding affinity to lactogenic sites could not be related to receptor occupancy degree. It is proposed that binding of hGH:MAb AE5 complexes to receptors would elicit a conformational change on adjacent receptor molecules leading to an increase of their affinity to bind subsequent hGH:MAb AE5 complexes.

Identification of a linear epitope of interferon-alpha2b recognized by neutralizing monoclonal antibodies.

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-alpha2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.

Detection of negative allosteric effects between monoclonal antibodies by using an antigenic model-builder computer program.

The ability of monoclonal antibodies (MAb) to bind or not simultaneously to the antigen (Ag) is used to establish antigenic maps considering that two different MAb do not bind to the Ag when the corresponding epitopes are overlapped (steric effect). Nevertheless, MAb inducing negative allosteric effect on the Ag could prevent the binding of the second MAb even if it is directed to a separate epitope. We report here that a knowledge-based expert module included in our previously described antigenic model-builder program (MAPAG) was able to differentiate between steric and negative allosteric effects between some MAb.

Monoclonal antibodies to human growth hormone modulate its biological properties.

Previous results indicated that monoclonal antibodies (mAbs) termed mAb AE5, mAb AC8 and mAb F11, recognizing the human growth hormone (hGH) region left exposed after binding to lactogenic, somatogenic and hGH-specific receptors, produce allosteric changes in the hormone which modify its binding properties. To study whether these mAbs could also influence hGH biological activity, experiments were carried out with Nb2 cells, a rat lymphoma cell line which proliferates in the presence of lactogenic hormones. Experiments involving previous binding of the hormone to receptors before adding 125I-mAbs indicated that the hGH domain defined by overlapped epitopes AE5, AC8 and F11 is uncovered in hGH when it is bound to the cell membranes. To reveal any alteration in the hGH molecule induced by the mAbs, preformed 125I-mAb:hGH complexes were added to the cell membranes. Data showed that 125I-mAb AE5:hGH complexes bound better to the receptors than free hormone. On the contrary, hGH previously bound to 125I-mAb AC8 or 125I-mAb F11 was poorly recognized by Nb2 receptors. Furthermore, both mAbs AC8 and F11 strongly inhibited 125I-hGH binding to Nb2 cell membranes and hGH-induced Nb2 cell proliferation whereas mAb AE5 enhanced both hormone binding and hGH mitogenic effect. Additionally, since mAb AC8 is directed towards an epitope shared by hGH and human placental lactogen (hPL), it was also shown that this mAb could impair hPL biological activity even though it recognizes the hPL region left exposed in hPL:Nb2 cell receptor complexes. Data presented in this work suggest that mAbs directed to the hGH or hPL regions unmasked after binding to Nb2 cell receptors produce allosteric alterations in the binding properties of these hormones leading to either enhancement or decrease of their biological activities.

MAPAG: a computer program to construct 2- and 3-dimensional antigenic maps.

The contact area between an antibody (Ab) and the antigen (Ag) is called antigenic determinant or epitope. The first step in the characterization of an Ag by using monoclonal antibodies (MAb) is to map the relative distribution of the corresponding epitopes on the Ag surface. The computer program MAPAG has been devised to automatically construct antigenic maps. MAPAG is fed with a binary matrix of experimental data indicating the ability of paired MAb to bind or not simultaneously to the Ag. The program is interactive menu-driven and allows the user an easy data handling. MAPAG utilizes iterative processes to construct and to adjust the final map, which is graphically shown as a 2- or a 3-dimensional model. Additionally, the antigenic map obtained can be optionally modified by the user or readjusted by the program. The suitability of MAPAG was illustrated by running experimental data from literature and comparing antigenic maps constructed by the program with those elaborated by the investigators without the assistance of a computer. Furthermore, since some MAb could present negative allosteric effects leading to misinterpretation of data, MAPAG has been provided with an approximate reasoning module to solve such anomalous situations. Results indicated that the program can be successfully employed as a simple, fast and reliable antigenic model-builder.

Allosteric effects of monoclonal antibodies on human growth hormone.

We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors. The effect of MAbAE5,AC8 and F11 on hGH binding was measured by determining the formation of 125I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding 125I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes. Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case performed 125I-MAb:hGH complexes were added to microsomes. Data showed that 125I-MAb AE5:hGH complexes bound better to the various receptors than 125I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to 125I-MAb AC8 or 125I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32-46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding. Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies as probes to study the human growth hormone-binding domain to lactogenic rat liver receptors.

A set of monoclonal antibodies (MAb) to human GH (hGH) was used to study the hormone binding orientation to its receptors (R) from female rat liver. The hGH antigenic region left exposed after its binding to liver microsomes was detected by measuring the ability of various [125I]MAb to bind to the preformed hGH-R complexes. Results indicated that a cluster of epitopes defined by the MAb, termed AE5, AC8, and AE12, remains accessible in the hGH-R complex whereas overlapping epitopes 3C11 and HG3 would define a hGH region involved in the binding site. Supporting these findings, solubilization and HPLC gel filtration of [125I]MAb-hGH-R complexes showed a radioactive peak of about 450,000 mol wt for MAb AE5 or AC8, but not for MAb 3C11 or HG3. [125I]MAb AE12 behaved differently, suggesting that epitope AE12 may be masked or altered in hGH-R-solubilized complexes. MAb directed to the putative hGH-binding site (MAb 3C11, HG3, and the closely related MAb 10C1 and NA71) failed to inhibit binding of the preformed [125I]MAb AE5-hGH complex to the receptors, suggesting a hormone modification after MAb AE5 binding. Accordingly competition experiments indicated an increase in the affinity of hGH for its receptors induced by this MAb. A higher hGH concentration was required to obtain 50% [125I]hGH binding to liver microsomes in the presence of MAb AE5 than in its absence. As the MAb used define epitopes that were previously correlated with the hGH structure, we concluded that a high flexible region (sequences 134-150) is exposed in the hGH-R complex. Furthermore, some MAb directed to this region enhance the hormone affinity for its rat liver receptors, probably through an induced conformational change.

Preparation of 125I-labeled human growth hormone of high quality binding properties endowed with long-term stability.

125I-labeled human growth hormone (125I-labeled.hGH) was prepared by using two variants of the chloramine T labelling procedure and purified by polyacrylamide gel electrophoresis (PAGE) of the reaction mixture. Variant A produced a tracer with high specific activity (100 +/- 10 microCi/microgram), high maximal binding capacity to antibodies (93%) and long-term stability (at least 150 days at -20 degrees C). No diiodinated tyrosil residues could be detected in this tracer. Variant B was devised to obtain higher yields of labeled hormone. The electrophoresis of the iodination mixture revealed two radioactive components with Rm values of 0.49 and 0.55 which result from the iodination of hGH variants preexisting in the starting material. Both tracers had similar specific activities (70 +/- 10 microCi/microgram), high maximal binding capacity to antibodies or receptors (80-100%, after 80 days of their obtention) and high stability (at least 100 days at -20 degrees C). It is concluded that the iododerivatives of hGH obtained by either method are adequate to perform radioimmunoassay and receptor studies and have long-term stability.

Binding in vitro to rat liver receptors does not correlate with activities in vivo of bovine somatotropin. Use of chemically modified derivatives as probes.

Bovine somatotropin with an increasing number of its carboxylate groups modified by reaction with glycine methyl ester in the presence of a water-soluble carbodi-imide was tested for its activity in different bioassays. Only those derivatives which were known to be active in the body-weight-increase bioassay were able to compete with 125I-labelled bovine somatotropin for their specific binding sites in vivo. No difference was found in the rate of clearance of a poorly active derivative as compared with that of native somatotropin. In contrast, both active and inactive derivatives were found to be equally effective in displacing the tracer from its binding sites present in isolated cells and membrane preparations from rat liver. These results suggest that the liver somatogenic receptors studied in vitro are less discriminating than those detected in vivo.

Formation of complexes between 125I-labelled human or bovine somatotropins and binding proteins in vivo in rat liver and kidney.

At 5 min after intravenous injection, both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin were concentrated in rat liver and kidney. When the labelled hormones were administered along with an excess of the corresponding unlabelled hormone, a significant decrease of the uptake was observed in the liver, but not in the kidney. Study of the subcellular distribution of radioiodinated somatotropins in liver revealed that most of the radioactivity was specifically concentrated in the microsomal fraction. In contrast, the kidney fraction that accounted for most of the radioactivity was the 100 000 g supernatant. After solubilization, with 1% (w/v) Triton X-100, of the microsomal fractions obtained from both organs, the radioactive material was analysed by gel filtration on Sepharose CL-6B. By using this approach, it was demonstrated that both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin bind in vivo to proteins present in liver. A small proportion of 125I-labelled human somatotropin was also shown to form complexes with proteins present in kidney. The present results demonstrate that the liver uptake is mainly due to binding of somatotropins to specific proteins, in contrast with the kidney, in which binding to specific sites contributes minimally to the overall uptake.

Properties of human growth hormone binding sites solubilized from female rabbit kidney.

Human growth hormone binding sites from female rabbit kidney microsomes were solubilized by treatment with the nonionic detergent Triton X-100. The binding of 125I-labelled human growth hormone to the solubilized sites retains many of the properties observed in the particulate fraction, such as saturability, reversibility, high affinity and structural specificity. The association and the dissociation process are time- and temperature-dependent. The association rate constant, k1, is 1.6 . 10(7) mol-1 . 1 . min-1 at 25 degrees C, and the dissociation rate constant, k-1, is 2.8 . 10(-4) min-1 at 25 degrees C. Solubilization causes an increase in affinity as well as in binding capacity. Scatchard plots from saturation curves suggest the presence of a single class of binding sites with a dissociation equilibrium constant, Kd, of 1.3 . 10(-11) M and a binding capacity of 133 fmol/mg of protein. Similar results were obtained from competition experiments. Specificity studies revealed the lactogenic characteristics of the solubilized sites. The Stokes radii of the free binding sites and of the 125I-labelled human growth hormone-binding site complex, determined on a Sepharose CL-6B column, are 57 and 53 A, respectively.