Significant Specific Binding Research Articles

Species differences in characteristics and distribution of [ 3H]α,β-methylene ATP binding sites in urinary bladder and urethra of rat, guinea-pig and rabbit

The characteristics and distribution of [ 3H]α, β-methylene ATP ([ 3H]α, β-MeATP, a radioligand for P 2x-purinoceptors) binding sites in the urinary bladder of rat, guinea-pig and rabbit were examined. Autoradiographic localization of [ 3H]α, β-MeATP binding sites was also carried out on the urethra of the three species. Receptor binding assay showed that the rat bladder possesses the highest density of specific binding sites, followed by rabbitand guinea-pig bladder. Semi-quantitative analysis of autoradiograms showed that the grain densities in the bladders of rat, guinea pig, and rabbit were parallel to those obtained from receptor binding assay. The grain densities were greatly reduced in the presence of β,γ-methylene ATP (β,γ-MeATP). No significant specific binding was detected in the smooth muscle of rat and guinea-pig urethra, while a very low level of specific binding was observed in the rabbit urethra. Differences of grain densities different regions (dome, body, and trigone) of the same bladder were also observed, but they were not as remarkable as those between species. The results of this study demonstrate species differences of P 2x-purinoceptor densities in the urinary bladder, which may reflect differing degrees of purinergic neurotransmitter control of the bladder detrusor muscle.

Myeloid and Erythroid Progenitor Cells From Normal Bone Marrow Adhere To Collagen Type I

One of the mechanisms by which normal hematopoietic progenitor cells remain localized within the bone marrow microenvironment is likely to involve adhesion of these cells to extracellular matrix (ECM) proteins. For example, there is evidence that uncommitted, HLA-DR-negative progenitor cells and committed erythroid precursors (BFU-E) bind to fibronectin. However, fibronectin is not known to mediate binding of committed myeloid (granulocyte-macrophage) progenitors, raising the possibility that other ECM proteins may be involved in this process. We investigated the binding of the MO7 myeloid cell line to a variety of ECM proteins and observed significant specific binding to collagen type I (56% +/- 5%), minimal binding to fibronectin (18% +/- 4%) or to laminin (19% +/- 5%), and no binding to collagen type III, IV, or V. Similarly, normal bone marrow myeloid progenitor cells (CFU-GM) demonstrated significant specific binding to collagen type I (46% +/- 8% and 47% +/- 12% for day 7 CFU-GM and day 14 CFU-GM, respectively). The ability of collagen to mediate binding of progenitor cells was not restricted to the myeloid lineage, as BFU-E also showed significant binding to this ECM protein (40% +/- 10%). The binding of MO7 cells and CFU-GM was collagen-mediated, as demonstrated by complete inhibition of adherence after treatment with collagenase type VII, which was shown to specifically degrade collagen. Binding was not affected by anti-CD29 neutralizing antibody (anti-beta-1 integrin), the RGD-containing peptide sequence GRGDTP, or divalent cation chelation, suggesting that collagen binding is not mediated by the beta-1 integrin class of adhesion proteins. Finally, mature peripheral blood neutrophils and monocytes were also found to bind to collagen type I (25% +/- 8% and 29% +/- 6%, respectively). These data suggest that collagen type I may play a role in the localization of committed myeloid and erythroid progenitors within the bone marrow microenvironment.

Myeloid and erythroid progenitor cells from normal bone marrow adhere to collagen type I

One of the mechanisms by which normal hematopoietic progenitor cells remain localized within the bone marrow microenvironment is likely to involve adhesion of these cells to extracellular matrix (ECM) proteins. For example, there is evidence that uncommitted, HLA-DR-negative progenitor cells and committed erythroid precursors (BFU-E) bind to fibronectin. However, fibronectin is not known to mediate binding of committed myeloid (granulocyte-macrophage) progenitors, raising the possibility that other ECM proteins may be involved in this process. We investigated the binding of the MO7 myeloid cell line to a variety of ECM proteins and observed significant specific binding to collagen type I (56% +/- 5%), minimal binding to fibronectin (18% +/- 4%) or to laminin (19% +/- 5%), and no binding to collagen type III, IV, or V. Similarly, normal bone marrow myeloid progenitor cells (CFU-GM) demonstrated significant specific binding to collagen type I (46% +/- 8% and 47% +/- 12% for day 7 CFU-GM and day 14 CFU-GM, respectively). The ability of collagen to mediate binding of progenitor cells was not restricted to the myeloid lineage, as BFU-E also showed significant binding to this ECM protein (40% +/- 10%). The binding of MO7 cells and CFU-GM was collagen-mediated, as demonstrated by complete inhibition of adherence after treatment with collagenase type VII, which was shown to specifically degrade collagen. Binding was not affected by anti-CD29 neutralizing antibody (anti-beta-1 integrin), the RGD-containing peptide sequence GRGDTP, or divalent cation chelation, suggesting that collagen binding is not mediated by the beta-1 integrin class of adhesion proteins. Finally, mature peripheral blood neutrophils and monocytes were also found to bind to collagen type I (25% +/- 8% and 29% +/- 6%, respectively). These data suggest that collagen type I may play a role in the localization of committed myeloid and erythroid progenitors within the bone marrow microenvironment.

Evidence for the expression of growth hormone receptors in human placenta

Since human placenta produces a growth hormone variant, it seemed important to search for evidence of GH receptors in that organ. Evidence for the expression of the GH receptor (GHR) gene was obtained by northern blot analysis. In addition, GHR poly A+RNA was detected in RNA from cultured trophoblastic cells, but not from placenta fibroblasts. There was a low but significant specific binding of pituitary GH-N and placental GH-V to placenta plasma membranes. Both variants apparently bound to the same receptor, which is present in the first trimester as well as in the term placenta. These results suggest that placental GH may have paracrine or autocrine functions in the placenta.

Phylogeny of tachykinin receptor localization in the vertebrate central nervous system: apparent absence of neurokinin-2 and neurokinin-3 binding sites in the human brain

Binding of [ 125I]Bolton-Hunter labeled tachykinins substance P (BHSP), neurokinin A (BHNKA) and eledoisin (BHELE) to brain sections from several vertebrates was investigated by receptor autoradiography. Densities of BHSP binding sites were low in fish brain, increased in lower vertebrates, were high in birds and rodents, and relatively constant in cat, monkey and human. In constant, BHELE binding site densities were moderate in fish brain and high in frog, snake, chick, pigeon, mouse and rat brain. Low and very low densities were localized in guinea pig and cat, while no significant BHELE specific binding was found in monkey and human brain. BHSP and BHELE binding sites were distinctly distributed in the vertebrate brains analyzed. Each ligand showed a characteristic regional distribution which was similar from species to species. The affinity profiles of tachykinins for BHSP and BHELE binding sites as analyzed on frog, chick and rat brain sections, corresponded to the NK1 and NK3 receptor types, respectively. No BHNKA binding sites could be detected in any vertebrate brain investigated. In conclusion, marked species variations exist in the density and distribution of tachykinin receptor types in the vertebrate brain. Thus, neurokinin A receptors (NK2 type) seem to be absent in the vertebrate central nervous system and, while substance P receptors (NK1 type) appear to be preserved and increase in density during evolution, the contrary seems to happen for the eledoisin receptors (NK3 type) which are more abundant in lower vertebrates and apparently absent in primate, particularly human brain.

Topographical distribution of 5-HT3 receptor recognition sites in the ferret brain stem

The distribution of [3H]zacopride (1.0 nM) to putative 5-HT3 receptor recognition sites in the ferret hindbrain was assessed using autoradiography. Specific binding (defined by the inclusion of granisetron, 1.0 microM) was heterogeneously distributed with highest density within the dorsal vagal complex (area postrema, nucleus tractus solitarius and dorsal motor nucleus of the vagus nerve). Lower densities were detected in the spinal trigeminal nerve complex whilst no other significant specific binding was detected ventral to the dorsal vagal complex. The location of 5-HT3 receptor recognition sites within the dorsal vagal complex may provide sites of action for zacopride and other 5-HT3 receptor antagonists to inhibit the emesis induced by cancer chemotherapeutic agents and x-radiation.

Autoradiographic analysis of the distribution of vasoactive intestinal peptide binding sites in the vertebrate central nervous system: a phylogenetic study

The distribution of vasoactive intestinal peptide (VIP) binding sites in the brain of several vertebrate species was examined by in vitro autoradiography on slide-mounted sections. This study included fish, frog, snake, pigeon, rat, mouse, guinea pig, cat and monkey brain. A fully characterized, monoiodinated form of vasoactive intestinal peptide (M- 125I-VIP), which maintains the biological activity of the native peptide in the central nervous system (CNS), was used throughout the study. Among the lower vertebrate species, no significant specific binding was found in the fish brain, whereas in the frog and snake brain, specific VIP binding sites were observed, mainly in the telencephalon. In the pigeon brain, high densities of VIP binding sites were localized in the hyperstriatum, neostriatum, archistriatum, hippocampal area, dorsolateral cortical area and in the optic tectum. Ectostriatum and paleostriatum augmentatum displayed lower densities of specific binding. In mammals, the highest concentrations of VIP binding sites were observed in the rodent brain. In the rat, mouse and guinea pig brain, high densities were detected in the olfactory bulb, external layers of the cerebral cortex, dentate gyrus, midline thalamic nuclei, geniculate nuclei, some hypothalamic nuclei, superior colliculus coeruleus. Intermediate densities were found in amygdala, caudate-putamen, septum and nucleus accumbens, CA1–CA3 fields of the hippocampus and central gray. The cerebellum of these species presented high densities of VIP binding sites, with species to species differences in their localization. The non-specific binding was, however, increased in the rodent cerebellum. Lower densities of VIP binding sites were observed in the cat and monkey CNS. In these two species, the non-specific binding was considerably higher than in the lower mammals brain. In the cat and monkey brain, as in the lower mammals, the highest densities were revealed in the neocortex, dentate gyrus, thalamic nuclei and some midbrain structures including substantia nigra and locus coeruleus. In all the species studied, the white matter was never labeled with M- 125I-VIP. This study suggests that VIP binding sites appear relatively early in the evolution of the vertebrate CNS. The most important densities of specific VIP binding sites are observed in the pigeon and rodent brain, whereas the cat and monkey present a marked increase in non-specific binding. It is interesting to note that the distribution of VIP binding sites as revealed by autoradiography is quite conservative in terms of evolution and indicates an association, although non-exclusive, of VIP receptors with brain regions involved in the processing of specific sensory inputs.

Binding of human factors X and Xa to HepG2 and J82 human tumor cell lines. Evidence that factor Xa binds to tumor cells independent of factor Va.

Previous studies demonstrated that several cultured human tumor cell lines potentiate the conversion of prothrombin to thrombin by factor Xa and calcium in the absence of exogenous factor Va. In the present study, the specific binding of radioiodinated preparations of human factor X and factor Xa to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule, and a human bladder carcinoma cell line (J82) that does not synthesize factor V/Va, was examined. Radioiodinated factor Xa bound specifically to J82 and HepG2 cells, whereas no significant specific binding of 125I-factor X to either cell was observed. The binding isotherm of 125I-factor Xa to each tumor cell line exhibited a hyperbolic profile, and Scatchard analysis demonstrated a single class of binding site for factor Xa on each cell surface with Kd values of 1.66 +/- 0.39 and 1.64 +/- 0.52 nM and 566,000 +/- 71,000 and 28,000 +/- 6,000 binding sites/cell for HepG2 and J82 cells, respectively. Thrombin formation by cell-bound factor Xa was hyperbolic and saturable at 5 nM factor Xa on each cell line. Hanes-Woolf plots of the prothrombin activation data indicated that half-maximal rates of thrombin formation occurred at factor Xa concentrations of 1.50 +/- 0.43 nM and 1.42 +/- 0.48 nM on HepG2 and J82 cells, respectively. Pretreatment of J82 cells with polyclonal anti-human factor V IgG had no measurable effect on either the binding of 125I-factor Xa or prothrombin activation. However, pretreatment of HepG2 cells with anti-human factor V IgG inhibited prothrombin activation in a dose-dependent manner, but did not inhibit the binding of factor Xa to this cell. When both cell lines were preincubated with exogenous human factor Va, the binding of factor Xa to either HepG2 or J82 cells was marginally affected. These data indicate that HepG2 and J82 cells have cell surface factor Xa binding sites proximal to, but independent of, cell surface factor Va of either exogenous or endogenous origin.

VirG, an Agrobacterium tumefaciens transcriptional activator, initiates translation at a UUG codon and is a sequence-specific DNA-binding protein

The Agrobacterium tumefaciens Ti plasmid virG locus, in conjunction with virA and acetosyringone, activates transcription of the virulence (vir) genes. Insertional and deoxyoligonucleotide-directed mutagenesis studies showed that both octopine and nopaline Ti plasmid virG genes initiate translation at a UUG codon. VirG protein initiated at this UUG codon was found to be 241 amino acid residues in length and had an apparent molecular mass of 27.1 kilodaltons. A Salmonella typhimurium trp-virG transcriptional fusion was constructed to overproduce VirG. Agrobacterium cells containing this gene fusion showed a large increase in virG activity in the presence of virA and acetosyringone. Since the trp promoter is not under virA-virG control, this result indicates that modification of VirG is necessary for its full activity. VirG overproduced in Escherichia coli was purified from inclusion bodies. It was found to be a DNA-binding protein that preferentially bound DNA fragments containing the 5' nontranscribed regions of the virA, -B, -C, -D, and -G operons. Significant specific binding to the 5' nontranscribed region sequences of virE was not detected. DNase I footprinting of the upstream regions of virC-virD and virG showed that VirG binds to sequences around the vir box region.

HLA class II-restricted binding of muramyl peptides to B lymphocytes of normal and narcoleptic subjects

The propensity of narcolepsy, a clinical sleep disorder of unknown etiology, is virtually totally included within the HLA-DR2, DQw1 (DRw15, Qw6) phenotype. The disorder is characterized by decreased sleep latency, early onset of rapid eye movement sleep, and a paucity of noctural slow-wave sleep. Muramyl peptides, naturally occurring bacterial cell wall peptiodoglycans, potently enhance the duration and amplitude of slow-wave sleep in animals, bind to murine monoclear r cells, and exhibit a major histocompatibility complex-restricted immunoadjuvant effect in mice. We examined the binding of muramyl peptides to peripheral blood mononuclear leukocytes of HLA-typed normal ( n = 13) and narcoleptic ( n = 10) sunjects. Muramyl peptides bound specifically and with high affinity to normal B-butt not T-lymphocyte-enriched preparations. There was no significant specific binding to B-cell-enriched preparations from narcoleptic patients. Furthermore, B-lymphocyte-enriched preparations of normal individuals who had the HLA-DR2,DQw1 phenotype ( n = 8) exhibited a lower level of specific binding than those of normals who did not have this phenotype ( n = 5, p < 0.001). These observations are an additional indication of the relevance of muramyl peptides to slow-wave sleep and provide a basis for a better understanding of the relation between narcolepsy and the MHC at the biochemical level.

3H]AHN 086 acylates peripheral benzodiazepine receptors in the rat pineal gland

AHN 086, an isothiocyanato derivative of Ro 5-4864 (4′-chlorodiazepam), inhibits radioligand binding to peripheral benzodiazepine receptors with characteristics of an irreversible (acylating) ligand. We now report that [ 3H]AHN 086 labels a ≈ 30 kDa protein in the rat pineal gland determined by both SDS-polyacrylamide gel electrophoresis and gel filtration high-performance liquid chromatography of digitonin-solubilized membranes. Specific incorporation of [ 3H]AHN 086 into this protein was inhibited by preincubating membranes with excess AHN 086. Moreover, significant specific binding of [ 3H]AHN 086 was not observed in either bovine pineal gland (which does not possess high-affinity binding sites for Ro 5-4864) or ovalbumin. These findings suggest that the ≈ kDa protein labeled by [ 3H]AHN 086 in rat pineal gland is associated with peripheral benzodiazepine receptors in this tissue.

Species variations in 5-HT3 recognition sites labeled by 3H-quipazine in the central nervous system.

The specific binding of 3H-quipazine to putative 5-HT3 receptors was analyzed in multiple species. Specific and saturable binding of the radioligand could be detected in both rat (KD = 1.2 nM; Bmax = 3.0 pmol/g) and pig (KD = 1.3 +/- 0.2 nM; Bmax = 1.5 +/- 0.2 p/mol/g) cortical membranes. By contrast, no significant specific binding of 3H-quipazine could be detected in human, cow, dog, turtle, mouse, guinea pig, chicken or rabbit brain membranes. These data indicate that marked species variations exist in the presence and/or density of 5-HT3 membrane recognition sites in the central nervous system.

Effect of dietary restriction on total and bacterium-specific mucosal secretory immunoglobulin A in bile-diverted intestinal self-filling blind loops.

The effect of starvation on the mucosal secretory immunoglobulin A (sIgA) response to bacterial antigens was studied in bile-free rat self-filling blind loops constructed at the end of a Roux-en-Y branch of jejunum. Rats were fed a 50% restricted diet for 1 to 4 weeks after surgery. sIgA was measured in the mucosa and lumen by an enzyme-linked immunosorbent assay. Dietary restriction caused a final rise of luminal sIgA which was less than 50% of that of normally fed controls Luminal bacteria counts were not different in the two groups. The percentage of total sIgA precipitated with intestinal bacteria was not significantly affected by dietary restriction, and there was no change in the specific binding of sIgA to several bacterial species. Nonprecipitated sIgA exhibited a low but significant specific binding to bacteria in both diet-restricted and fed rats. Diet restriction therefore reduced the total sIgA response to luminal bacteria, but the specific bacterial binding capacity per microgram of sIgA was not altered. In these short-term experiments diet-restricted animals appeared to be capable of secreting sIgA in excess of requirement, since the nonprecipitable luminal fraction contained free sIgA with binding capacity for bacteria. The ability of sIgA to react with specific antigens may therefore be of more significance as an indicator of bacterial susceptibility than the measurement of total sIgA.

The effect of hCG on cyclic AMP in rabbit ciliary processes.

Experiments designed to address the role of cyclic AMP as a mediator of the eye pressure and aqueous flow reducing effects of human chorionic gonadotropin (hCG) in the rabbit have been conducted. No significant stimulatory effect of hCG on particulate preparations of adenylate cyclase from rabbit ciliary processes could be found under several different experimental conditions. Forskolin, an activator of adenylate cyclase can, in some biological systems, reveal a significant hormonal cyclic AMP response which is undetectable in the presence of hormone alone. Forskolin did potentiate the effect of isoproterenol on cyclic AMP production by intact rabbit ciliary processes. Cyclic AMP production by intact ciliary processes was not affected by hCG, either in the presence or absence of forskolin. Significant specific binding of iodinated hCG to particulate preparations of rabbit ciliary processes could not be detected. These results dispute the role of cyclic AMP as a mediator of the flow and pressure reducing effects of hCG; however, theoretical and methodological complications relating to this interpretation are discussed.

Characterization of hepatic growth hormone binding sites in two fish species, Gillichthys mirabilis (teleostei) and Acipenser transmontanus (chondrostei)

To obtain information on the presence of growth hormone (GH) receptors in liver of nonmammalian vertebrates the specific binding of 125I-bovine growth hormone (bGH) to liver membranes of seven species representing the major groups was studied by radioreceptor assay. A substantial degree of specific binding was detected with sturgeon ( Acipenser transmontanus) liver membranes and a much lower amount was detected on hepatic membranes of Gillichthys mirabilis. No significant specific binding was detected on liver membranes of pigeon, turtle, bullfrog, tilapia, or leopard shark. Gillichthys and sturgeon liver membranes were further characterized and compared with hepatic membranes from male rabbits. The sturgeon and Gillichthys membranes showed binding that was dependent upon time, temperature, pH, and membrane concentration. Scatchard analysis of the binding of 125I-bGH to sturgeon and rabbit membranes revealed both high and low affinity binding sites. The high affinity sites had K A values of 3.1 × 10 11 and 1.0 × 10 11 M −1, and capacities of 12 and 50 fmol/mg protein, respectively. Membranes from Gillichthys liver contained only a single class of binding sites with a K A of 6.7 × 10 9 M −1 and a binding capacity of 49 fmol/mg. Hormonal specificity of the sturgeon and Gillichthys hepatic binding sites was studied using methionyl-human GH (met-hGH), ovine prolactin (oPRL), and a crude preparation of sturgeon (st)GH. The met-hGH and stGH inhibited the binding of 125I-bGH to sturgeon liver membranes while only met-hGH displaced labeled bGH from Gillichthys liver membranes. One μg of oPRL did not significantly inhibit 125I-bGH binding in either membrane assay. Based on these studies, sturgeon hepatic GH receptors seem to be more like those of nonprimate mammals than those of teleosts. Our results, in conjunction with the data of J. N. Fryer ( Gen. Comp. Endocrinol. 39, 123–130 (1979)), indicate that considerable evolutionary divergence has occurred among teleost hepatic GH receptors. Thus, vertebrate GH receptors seem to have undergone at least as much evolution as has the hormone itself.

Hormone receptors in the epiphysial cartilage.

In order to assess which hormones may exert direct effects on skeletal growth at the epiphysial growth plate, the specific binding of hormones to the epiphysial cartilage of growing dogs and rabbits was studied. Membrane fractions obtained by centrifugation of homogenates prepared from dog and rabbit growth plate cartilage at 600, 15 000 and 105 000 g showed significant specific binding of serum insulin-like activity and insulin. Binding of growth hormone and prolactin by the three membrane fractions was negligible. Saturable binding sites for triiodothyronine could be demonstrated in nuclei from the dog growth plate. Nuclear binding showed an apparent Kd of 11 +/- 3.6 nmol/l and a maximum binding capacity of 4.1 +/- 1.6 pmol/mg DNA, a level comparable to dog liver. Using a viable chondrocyte suspension prepared from dog epiphysial cartilage, specific steroid binding in the cells could be demonstrated for [3H]dexamethasone but not 17 alpha-methyltrienolone, oestradiol-17 beta or 1 alpha, 25-dihydroxycholecalciferol. Scatchard analysis of dexamethasone binding showed high affinity binding sites having a Kd of 1.2 +/- 0.35 nmol/l and a capacity of 1700 sites/cell, and a low affinity binding with a Kd of 109 +/- 57 nmol/l and a capacity of 24 000 sites/cell. Steroid competition for the specific binding showed the following sequence of affinity: dexamethasone greater than corticosterone greater than 11-deoxycortisol greater than testosterone greater than oestradiol-17 beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated B cells express receptors for, and proliferate in response to, pure interleukin 2.

In this study we investigated whether interleukin 2 (IL-2) acts on B cell proliferation and whether activated B cells express IL-2 receptors. First, the functional activity of immunoaffinity-purified or recombinant human IL-2 was studied in a B blast assay using positively selected murine surface Ig-positive cells that had been activated by lipopolysaccharide (LPS) plus anti-Ig antibodies (anti-Ig). In this assay, T cells were not detected by fluorescence-activated cell sorter analysis. It was found that both IL-2 preparations led to optimal B cell proliferation compared with supernatants obtained from murine or human spleen cells or murine cloned T helper cells. Second, we observed that the IL-2 requirement in this assay was about the same as in a proliferation assay using lectin-activated polyclonal murine Lyt-2-positive T cells. Third, analysis of the binding of radiolabeled immunoaffinity-purified IL-2 to B cells indicated that LPS plus anti-Ig-activated B cells expressed a mean of 3,500 IL-2 receptors per cell with an apparent dissociation constant of 150 pM. However, neither nonactivated B cells nor B cells activated by LPS alone exhibited significant specific IL-2 binding. The functional and the receptor data are consistent with the conclusion that IL-2 is a growth factor not only for T cells but also for B cells.

Specific binding of a stable prostacyclin analogue (Iloprost) to rat oxyntic mucosa

It has been reported that prostacyclin (PGI 2) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI 2 is inactivated under physiological conditions it has not been possible to demonstrate specific PGI 2 binding to the rat stomach. Therefore a stable PGI 2 analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE 2 nor 6 keto PGF 1α displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 × 10 −11 M and 7.1 × 10 −8 M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI 2 was less potent than unlabelled Iloprost in displacing 3H-Iloprost from its binding site. The addition of PGE 2 to the incubation medium resulted in an increase in 3H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI 2-like agents but not for either PGE 2 or 6 keto PGF 1α.

Transformation of LMTK- cells with purified class I genes. V. Antibody-induced structural modification of HLA class I molecules results in potentiation of the fixation of a second monoclonal antibody.

A potentiation phenomenon was observed with HLA-A3 and CW3 transformed murine L cells between anti-HLA class I B10.6 (potentiated) and B10.8 (potentiating) monoclonal antibodies (m.Ab.). Further studies of this phenomenon with these transformed L cells indicated that: 1) no significant specific binding of B10.6 m.Ab. to HLA-A3 and CW3 transformed L cells could be demonstrated by conventional radioimmunoassay or cytofluorometric study in the absence of B10.8 m.Ab.; 2) potentiation of the fixation of B10.6 m.Ab. was induced by other anti-HLA class I m.Ab., which all reacted with the same cluster of antigenic determinants; 3) potentiation reflects an increased specific fixation of B10.6 m.Ab. to HLA class I molecules implicating its combining site; 4) potentiation was mediated by B10.8 Fab fragments. These results indicate that potentiation of the fixation of B10.6 m.Ab. to the HLA-A3 and CW3 molecules expressed by the transformed L cells reflects conformational changes of these molecules after interaction with B10.8 m.Ab.

Follitropin binding to receptors in testis: Studies on the reversibility and thermodynamics of the reaction

Studies were designed to assess the extent to which binding of follitropin (FSH) to its receptor is a reversible process. [ 125I]hFSH was allowed to interact with membrane receptors from calf testis for 2 h at various temperatures by which time significant specific binding of [ 125I]hFSH had taken place in all instances. Unlabeled FSH was then added (delayed addition) and the amount of [ 125I]hFSH remaining bound was monitored as a function of time. In order to assess reversibility, [ 125I]hFSH binding in the latter samples was compared to that occurring when unlabeled FSH had not been added or when unlabeled FSH was present from the start of the incubation. Binding was essentially fully reversible at 4°C, but reversibility decreased with increasing temperature. Reversibility of FSH binding decreased markedly at temperatures greater than 26°C and was considered irreversible at temperatures above 30°C. At 4°C essentially full reversibility (> 90%) was observed when the unlabeled hormone was added after 7 h of incubation, but decreased when added after 12 h. At warmer temperatures (22 or 30°C) there was a progressive decrease in reversibility as the time of delay before addition of unlabeled hormone was lengthened. By determining the affinity constant (in separate experiments) at various temperatures, a thermodynamic analysis was possible. This analysis was restricted to the temperature range 4–26°C in order to minimize complications arising from irreversible binding. The reaction was endothermal at low temperatures ( T < 12.5° C) and exothermal at higher temperatures ( T > 12.5° C) and was associated with a decrease in heat capacity of 1800 cal [mol deg] −1 at 25°C. The results are consistent with the concept that the hydrophobic effect plays an important role in FSH binding, but that the reaction is complex and may be composed of more than one step.