Biomedical subjects

L P Roguin

Publications and source records attributed to L P Roguin.

9 recordsLinked to original sources

PubMed1995

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.

Allosteric Regulation↗

PubMed1994

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)

Allosteric Regulation↗

PubMed1990

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.

Animals↗

PubMed1989

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.

Chloramines↗

PubMed1984

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.

Animals↗

PubMed1983

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.

Animals↗

PubMed1982

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.

Animals↗

PubMed1981

Human somatotropin binding to rabbit kidney microsomal fraction.

Specific binding of 125I-labelled human somatotropin was demonstrated in microsomal membranes (microsomes) from rat and rabbit kidneys. Female rabbit kidney microsomes showed the highest binding activity and were used for further study. The association of 125I-labelled human somatotropin was time- and temperature-dependent and the binding reaction was reversible. Scatchard analysis of saturation data indicated a dissociation equilibrium constant, KD, of 56 pM and a binding capacity of 37 fmol per mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the microsomes was specifically inhibited by hormones with lactogenic activity. The binding sites, as well as 125I-labelled human somatotropin, were not inactivated on incubation. Treatment of the microsomes with trypsin and chymotrypsin decreased the specific binding by over 90%. Preheating of the microsomes at 55 degrees C for 15 min abolished 50% of the specific binding activity.

Animals↗

PubMedDate not supplied

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.

Algorithms↗