Prostaglandin E2 binding site distribution and subtype classification in the rabbit iris-ciliary body.

The distribution and characteristics of specific binding sites for tritium labeled prostaglandin E2 (3H-PGE2) were examined in membrane preparations from rabbit iris-sphincter, iris and ciliary body. The majority of 3H-PGE2 specific binding sites were found in the ciliary body (46%) followed by the iris (37%) and the iris-sphincter muscle (5%). Scatchard analysis of saturable 3H-PGE2 binding sites in the ciliary body indicated a single binding site with a Kd of 2.81 nM and Bmax value of 84 fmoles bound/mg protein. Competition by agonists selective for the EP1, EP2 and EP3 receptor subtypes of the EP (PGE2) prostanoid receptor indicated that the majority of rabbit ciliary body 3H-PGE2 binding sites are of the EP2 subtype. Incomplete displacement of labeled 3H-PGE2 from its binding sites by the EP2 selective agonist 11-deoxy PGE1 suggests the presence of additional EP or non-EP binding sites. There was essentially no binding to EP1 receptor sites as defined by the displacement of 3H-PGE2 by 17-phenyl-trinor PGE2. A weak displacement of 3H-PGE2 by the EP3/EP1 specific agonist, sulprostone, may account for the presence of a small number of EP3 specific binding sites in this tissue. The predominant distribution of PGE2 binding sites in the ciliary body and their identification as EP2 selective, supports recent functional studies where topical application of prostanoids with EP2 but not EP1 or EP3 agonist activity resulted in breakdown of the blood-aqueous barrier.     

Specific 3H-PGE1 binding sites in rat ovary in estrous cycle and pregnancy.

Stimulation of cAMP synthesis by prostaglandins E series in the rat ovary is consistent with the presence of a prostaglandin receptor in this tissue. Prostaglandin binding sites with specificity for PGE1 in vitro incubation systems have been demonstrated in rat ovary slices and corpora lutea. The binding of 3H-PGE1 was progressively inhibited with increasing amounts of unlabelled PGE1 and PGE2. PGF2alpha inhibitory effect was markedly smaller than that of PGE. 3H-PGE1 binding to the ovary was higher in 3-day-old rats than in 5-day-old and adult animals, when the highest binding was present in estrus. The specific binding of 3H-PGE1 to rat corpora lutea (CL) decreased on days 11 and 13 of pregnancy and then gradually returned to the level found on day 1 during the second half of gestation. This binding of labelled prostaglandin during pregnancy has been studied in relation to the PGE1 stimulation of cAMP synthesis in rat corpora lutea, but no consistent changes were observed in responsiveness.     

Modulation of 3H-prostaglandin E2 binding sites in the rabbit gastric mucosa

The binding of 3H-prostaglandin E2 (PGE2) to rabbit gastric mucosa was investigated. Binding depended on incubation time, temperature and pH, and was saturable and reversible. Scatchard plot analysis revealed a single class of binding sites with a dissociation constant (Kd) of 5.33 +/- 0.21 nM and a maximum number of binding sites (Bmax) of 138.1 +/- 3.4 fmol/mg protein. PGE1 and 16,16-dimethyl PGE2 potently competed with 3H-PGE2 for the binding sites of gastric mucosa, whereas PGA2, PGF2 alpha, 6-keto PGF1 alpha and thromboxane B2 were less potent. The gastric mucosa prepared from the rabbits given indomethacin (5 mg/kg s.c. three times) showed a lower Kd (2.47 +/- 0.19 nM) for 3H-PGE2 than that from untreated one. Treatment with a PGE1 analog, misoprostol (320 micrograms/kg s.c. three times) lowered the Bmax to 74.1 +/- 2.4 fmol/mg protein without any significant effect on the Kd value. It is concluded that rabbit gastric mucosa has specific binding sites for 3H-PGE2 which may be modulated by the levels of PGs in vivo.     

Demonstration of receptor heterogeneity and affinity modulation by nonequilibrium binding experiments. The cell surface cAMP receptor of Dictyostelium discoideum

The binding of [3H]cAMP to Dictyostelium discoideum cells was analyzed on a seconds time scale under both equilibrium and nonequilibrium conditions. The binding of [3H]cAMP increases rapidly to a maximum obtained at about 6 s, which is followed by a decrease to an equilibrium value reached at about 45 s. This decrease of [3H]cAMP binding is not the result of ligand degradation or isotope dilution by cAMP secretion but is due to a transition of high-affinity binding to low-affinity binding. Analysis of the dissociation rate of [3H]cAMP from the binding sites indicates that these high- and low-affinity binding sites are both fast dissociating with a half-life of about 1 s. In addition, these dissociation experiments reveal a third binding type which is slowly dissociating with a half-life of about 15 s. The number and affinity of these slowly dissociating sites does not change during the incubation with [3H]cAMP. The drugs caffeine and chlorpromazine do not change the total number of binding sites, but they change the ratio of the three binding types. In the presence of 10 mM caffeine almost all binding sites are in the low affinity conformation, while in the presence of 0.1 mM chlorpromazine the ratio is shifted to both the high-affinity type and slowly dissociating type. The results indicate that the cAMP-binding activity of D. discoideum cells is heterogeneous. In the absence of cAMP about 4% of the sites are slowly dissociating with Kd = 12.5 nM, about 40% are fast dissociating with high affinity (Kd = 60 nM), and about 60% are fast dissociating with low affinity (Kd = 450 nM). During the binding reaction the number of slowly dissociating sites does not change. The number of high-affinity sites decreases to a minimum of about 10% with a concomitant increase of low-affinity sites to about 90%. This transition of binding types shows first-order kinetics with a half-life of about 9 s. A half-maximal transition is induced by 12.5 nM cAMP.     

Release and specific binding of prostaglandins in bovine pineal gland

Incubated bovine pineal glands released prostaglandin E-and prostglandin F-like material (304 +/- 20 and 582 +/- 56 pg/mg dry tissue wt/h, respectively) and the release was increased 2.2 2.9-fold by adding 10(-4)-10(-6)M of norepinephrine to the medium. Binding assays revealed the existence of high affinity binding of 3H-prostaglandin E2 (3H-PGE2) and 3H-prostaglandin F1 alpha (3H-PGF2 alpha) in low speed supernatants of pineal homogenates. Binding was increased by increasing Ca++ concentration in medium up to 2 mM, was heat labile and was depressed following incubation with trypsin. In subcellular fractionation studies maximal 3H-PG binding was found in the 27000 x g pellet. Scatchard analysis of 3H-PGE2 binding revealed the presence of a single population of binding sites with a Kd= 1.2 nM and a binding site concentration of 1-2 pmoles/g protein. A single population of binding sites for 3H-PGF2 alpha was also detected with a Kd= 1.7 nM and a similar binding site concentration. Non-radioactive PGE1 and PGE2 were almost equally effective to compete for 3H-PGE1 binding sites (ED50= 5 and 2 nM, respectively). Unlabeled PGF1 was relatively ineffective to compete for 3H-PGE2 binding (ED50 greater than 1000 nM) but displaced effectively 3H-PGF2 alpha binding (ED20=1.2 nM).     

Pertussis toxin inhibits cAMP surface receptor-stimulated binding of [35S]GTP gamma S to Dictyostelium discoideum membranes

GTP-binding activity to Dictyostelium discoideum membranes was investigated using various guanine nucleotides. Rank order of binding activities was: GTP gamma S greater than GTP greater than 8-N3-GTP; the binding of GTP gamma S and GTP, but not of 8-N3-GTP, was stimulated by receptor agonists. [3H]GTP binding to D. discoideum membranes has been described previously by a single binding type (Kd = 2.6 microM, Bmax = 85 nM). More detailed studies with [35S]GTP gamma S showed heterogeneous binding composed of two forms of binding sites with respectively high (Kd = 0.2 microM) and low (Kd = 6.3 microM) affinity. cAMP derivatives enhanced GTP gamma S binding by increasing the affinity and the number of the high-affinity sites, while the low-affinity sites were not affected by cAMP. The specificity of cAMP derivatives for stimulation of GTP gamma S binding showed a close correlation with the specificity for binding to the cell surface cAMP receptor. Pretreatment of D. discoideum cells with pertussis toxin did not affect basal GTP and GTP gamma S binding, but eliminated the cAMP stimulation of GTP and GTP gamma S binding. These results indicate that D. discoideum cells have a pertussis toxin-sensitive GTP-binding protein that interacts with the surface cAMP receptor, suggesting the functional interaction of surface receptor with a G-protein in D. discoideum.     

Effect of topical application of estradiol-17 beta and PGE2 on PGE-binding sites in the porcine endometrium.

High- and low-affinity prostaglandin E2 (PGE2) binding sites were found on day 15 after estrus in the endometrium of cycling (Cy) and pregnant (Pr) gilts as well as gilts treated with intra-uterine Silastic beads containing estradiol-17 beta (E2) alone or in combination with PGE2 (E and PG gilts respectively) and inserted into the uterine lumen on day 10 of the cycle. The average apparent dissociation constants (Kd) and binding site concentrations (Bmax) for the high- and low-affinity sites were respectively (mean +/- SEM): 8.4 +/- 0.7 pM and 3.28 +/- 0.38 fmol/mg of protein and 5.3 +/- 0.8 nM and 71 +/- 9 fmol/mg of protein. Samples collected along the meso- and antimesometrial aspects did not differ (P greater than 0.05), although the low-affinity Bmax was higher on the antimesometrial aspect for Pr and Cy gilts only. No difference in Kd (P greater than 0.10) was found between treatments for high-affinity binding sites. For the low-affinity binding sites, Kd was higher for Pr compared to PG and E but not to Cy gilts (P less than 0.05). The high-affinity Bmax was higher (P less than 0.05) for PG, followed by E, Pr and Cy gilts (respectively: 5.50 +/- 0.26; 4.19 +/- 0.46; 1.78 +/- 0.40; 1.64 +/- 0.23 fmol/mg of protein), although Pr and Cy gilts were not different (P greater than 0.05). These results suggest that the localized presence of conceptuses in the uterus in early pregnancy does not markedly affect PGE binding sites but that intrauterine applications of Silastic beads containing E2 and PGE2 increase high-affinity Bmax and decrease low-affinity Kd.     

The existence of distinct classes of prostaglandin E2 receptors mediating adenylate cyclase and phospholipase C pathways in osteoblastic clone MC3T3-E1.

We have reported previously that PGE2 evoked an increase in intracellular calcium level ([Ca2+]i) in mouse osteoblastic cells (1). Here, we investigated the effects of PGE1 and PGF2 alpha on cAMP production and [Ca2+]i in comparison with those of PGE2. In osteoblastic clone, MC3T3-E1 cells, PGE1 stimulated cAMP production, but had no effect on [Ca2+]i, whereas PGF2 alpha evoked only [Ca2+]i increase. In contrast, PGE2 not only stimulated cAMP production, but also increased [Ca2+]i. From the Scatchard plot analysis of PGE2 it was confirmed that there were two classes of PGE2 binding sites (Kd value, 9.2 nM; binding site, 29 fmole/mg protein, and Kd value, 134 nM; binding site, 148 fmole/mg protein). As the increase in [Ca2+]i was caused by PGF2 alpha and PGE2, but not by PGE1, we investigated the displacement of [3H]-PGF2 alpha binding. The displacement capacity of unlabeled PGE2 was about 110 of that of PGF2 alpha, while that of PGE1 was very low even at 500-fold excess. These data indicate the possibility that the dual action of PGE2 is mediated by distinct receptor systems.     

High- and low-affinity interleukin 2 receptors: distinctive effects of monoclonal antibodies

We previously established several mouse hybridoma cell lines producing monoclonal antibodies against the human interleukin 2 (IL 2) receptor molecule. As they bind to both high- and low-affinity IL 2 receptors, their effects on binding of 125I-labeled IL 2 to high- and low-affinity receptors were examined by Scatchard plot analysis. Two of these monoclonal antibodies, HIEI and H-47, reduced the IL 2 binding affinity of high-affinity receptors from a Kd of 14 to 20 pM to a Kd of 110 to 140 pM, but slightly raised that of low-affinity receptors. These two antibodies scarcely affected the numbers of high- and low-affinity receptors. On the other hand, H-31 completely blocked IL 2 binding to both high- and low-affinity receptors, and H-A26 slightly reduced the affinities of both high- and low-affinity receptors, from 17 pM to 28 pM and from 28 nM to 54 nM, respectively. H-48 had little affect on IL 2 binding to high- or low-affinity receptors. By use of these monoclonal antibodies, the inhibitory effect of IL 2 on growth of an HTLV-I-immortalized T cell line was demonstrated to be transmitted from high-affinity, but not low-affinity, receptors.     

3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.     

Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin.

Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.     

Biological significance of phosphorylation and myristoylation in the regulation of cardiac muscle proteins

The effect of different dietary fats on peritoneal macrophage plasma membrane fluidity, intracellular cyclic AMP (cAMP) production, GTP hydrolysis and TNF binding and TNF-induced IL1 and IL6 production was investigated. After a four week period, fluidity, as determined by both fluorescence recovery after photobleaching (FRAP) and anisotropy was lowest and highest in animals fed corn and fish oil respectively. After eight weeks feeding, lateral membrane movements were decreased substantially in fish, olive and coconut oil fed dietary groups, whereas an increase in the corn oil fed group was observed, no effect was observed in macrophages from the butter fed group. However, an increase in the packing was observed in macrophages from all dietary groups except in the olive oil fed group. GTPase values for the coconut oil and butter groups were higher than in any other dietary group. After receiving the diet for 8 weeks these differences between the groups were no longer apparent. Exposure of macrophages to TNF had no effect on the rate of GTP hydrolysis. A major enhancement of cAMP production became apparent between weeks 4 and 8 of dietary treatment. After 4 weeks on the diet, values were significantly higher from cells of animals fed corn and olive oils than from animals fed fish oil. After 8 weeks, while there was a general enhancement of production, further differences became apparent. Feeding corn and coconut oils resulted in the highest values and olive oil and chow in the lowest. It is proposed that fats rich in n-3 fatty acids (fish oils) alter membrane fluidity, decrease TNF binding affinity, GTPase activity and cAMP production which appears not to modify cytokine production after short term dietary supplementation. However, after long term feeding it appears that increases in the sensitivity of the TNF receptors plays a major role in modifying cytokine production. (Mol Cell Biochem 166: 135-143, 1997)     

Binding studies and photoaffinity labeling identify two classes of phencyclidine receptors in rat brain

Binding and photoaffinity labeling experiments were employed in order to differentiate 1-(1-phenylcyclohexyl)piperidine (PCP) receptor sites in rat brain. Two classes of PCP receptors were characterized and localized: one class binds [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) with high affinity (Kd = 10-15 nM) and the other binds the ligand with a relatively low affinity (Kd = 80-100 nM). The two classes of sites have different patterns of distribution. Forebrain regions are characterized by high-affinity sites (hippocampus greater than frontal cortex greater than thalamus greater than olfactory bulb greater than hypothalamus), but some parts (e.g., hippocampus, hypothalamus) contain low-affinity sites as well. In the cerebellum only low-affinity sites were detected. Binding sites for [3H]PCP and for its photolabile analogue [3H]azido-PCP showed a regional distribution similar to that of the [3H]TCP sites. The neuroleptic drug haloperidol did not block binding to either the high- or the low-affinity [3H]TCP sites, whereas Ca2+ inhibited binding to both. Photoaffinity labeling of the PCP receptors with [3H]AZ-PCP indicated that five specifically labeled polypeptides of these receptors (Mr 90,000, 62,000, 49,000, 40,000, and 33,000) are unevenly distributed in the rat brain. Two of the stereoselectively labeled polypeptides (Mr 90,000 and 33,000) appear to be associated with the high- and low-affinity [3H]TCP-binding sites; the density of the Mr 90,000 polypeptide in various brain regions correlates well with the localization of the high-affinity sites, whereas the density of the Mr 33,000 polypeptide correlates best with the distribution of the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of nerve growth factor binding to cultured neural crest cells: evidence of an early developmental form of the NGF receptor

Cultured neural crest cells undergoing differentiation have been shown to contain a subpopulation of cells with specific receptors for nerve growth factor (NGF). These cells are the potential targets of NGF during differentiation and development. This study was done to pharmacologically characterize the binding of NGF to long-term (1- to 3-week) cultures of quail neural crest cells. The data indicate that 125I-NGF binding was specific and saturable, with less than 20% nonspecific binding. Scatchard analysis revealed the presence of one type (class) of receptors with a binding constant (Kd) similar to that of the low-affinity binding site described for embryonic dorsal root and sympathetic ganglia (approximately 3.2 nM). This was corroborated by displacement experiments (Kd of 1.3 nM), in which 125I-NGF binding was measured in the presence of increasing concentrations of nonradioactive NGF. In addition, affinity labeling revealed that the 125I-NGF-receptor complex had a molecular weight of about 93K, characteristic of the low-affinity NGF receptor of PC12 cells. The NGF receptor of cultured neural crest cells was trypsin-sensitive, as is typical of the low-affinity NGF binding sites. These findings indicate that differentiating neural crest cells lack high-affinity 125I-NGF binding sites. In contrast, embryonic dorsal root and sympathetic ganglia cells, known NGF targets, have both high- and low-affinity receptors. Measurements of the differential release of surface-bound 125I-NGF indicated that a relatively small amount (about 14%) of NGF is internalized over a 1-hr period. Cultured neural crest cells which bear NGF receptors were also shown by light microscopic radioautographic techniques to incorporate [3H]thymidine. I suggest, therefore, that cultured neural crest cells which have not terminally differentiated, as judged by morphological criteria and continued proliferation, may express an early developmental form of the NGF receptor.     

Early Undernutrition and [3H]γ-Aminobutyric Acid Binding in Rat Brain

The effect of early undernutrition and dietary rehabilitation on [3H]gamma-aminobutyric acid ([3H]GABA) binding in rat brain cerebral cortex and hippocampus was examined. Undernourished animals were obtained by exposing their mothers to a protein-deficient diet during both gestation and lactation. Saturation analysis of [3H]GABA binding in the cerebral cortex and hippocampus revealed high- and low-affinity components in the undernourished group, whereas control animals possessed only a low-affinity site. The concentration of low-affinity binding sites was greater in the undernourished animals. Rehabilitation of undernourished animals completely abolished the binding site differences. Treatment of brain membranes with Triton X-100 yielded two binding components in both the undernourished and control animals, although the concentration of lower affinity sites was still greater in the undernourished group. Neither the efficacy nor the potency of GABA to activate benzodiazepine binding in cerebral cortex was modified by undernutrition. These data suggest that early undernourishment modifies the characteristics of [3H]GABA binding, perhaps by reducing the brain content of endogenous inhibitors of the higher affinity binding site. The lack of effect on GABA-activated benzodiazepine binding suggests the possibility that neither the high- nor the low-affinity GABA binding sites are coupled to this receptor component.     

Prostaglandin E2 receptor heterogeneity and dysfunction in mammary tumor cells

We have reported previously that murine mammary tumor cell subpopulations isolated from one spontaneous adenocarcinoma are heterogenous in terms of prostaglandin E2 (PGE2) synthetic capacity. We have also shown that tumor-PGE2 contributes to the ability of these cells to grow and metastasize in vivo (Fulton and Heppner: Cancer Research 45:4779-4784, 1985). In the present study, we have asked whether exogenous PGE2 has direct effects on the proliferation of these cells in vitro and if such responses can be attributed to the capacity of these cells to 1) bind PGE2 and 2) activate adenylate cyclase via the PGE2 receptor. We report that PGE2, at concentrations below 1 x 10(-5) M, does not affect the proliferation rate of these cells. This unresponsiveness is not due to the absence of receptors for PGE2. However, marked heterogeneity in receptor binding and function was detected in these closely related cell lines. Two metastatic lines (66 and 410.4) have high-affinity receptors for PGE2 (average Kd = 4.3 x 10(-9) M/L and 4.2 x 10(-9) M/L, respectively) and similar binding capacities (4.1 x 10(-4) and 2.9 x 10(4) binding sites, respectively). Two nonmetastatic lines, 410 and 67, have receptors with lower affinity (Kd = 8.3 x 10(-9) M/L and 1.6 x 10(-7) M/L, respectively) and binding capacities of 2.8 x 10(5)/410 cell or 7.3 x 10(4)/67 cell. A third nonmetastatic line (168) exhibits no specific binding. PGE2 receptor stimulation leads to elevated intracellular cAMP in lines 66, 410, and 67. Line 410.4 cells appear to have a functional lesion in the PGE2 receptor resulting in a failure to elevate cAMP in response to receptor occupancy. Adenylate cyclase can, however, be activated in these cells by cholera toxin, NaF, or forskolin. In comparison to the other cell lines, line 168 cells respond poorly to all cAMP-stimulating agents. Thus, we have found that PGE2 binding is a heterogenous property for these cells, and, in addition, we have identified an apparent uncoupling of PGE2 receptor to the adenylate cyclase system in one cell line.     

Characterization of the guinea pig lung membrane leukotriene D4 receptor solubilized in an active form. Association and dissociation with an islet-activating protein-sensitive guanine nucleotide-binding protein

Membrane fractions from the guinea pig lung had high- and low-affinity binding sites for LTD4 with Kd values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or by prior treatment of the membrane with islet-activating protein (IAP), the high-affinity site shifted to a low-affinity state. Consistently, a 41-kDa protein was ADP-ribosylated by treatment of the lung membranes with IAP, and this event was inhibited by the addition of GTP gamma S. We solubilized the LTD4 receptor from the lung membranes in an active form with 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% glycerol. On a gel filtration column, the binding activity was eluted at the volume corresponding to a Mr of 70,000 or over 500,000 in the presence or absence of Mg2+ (5-20 mM), respectively, in solubilizing buffers. The Kd value of [3H]LTD4 binding to the 70-kDa protein was similar to the low-affinity binding constant of the membrane and was insensitive to GTP gamma S. The preparation solubilized in the absence of Mg2+ showed both high- and low-affinity binding sites for LTD4, and the addition of GTP gamma S shifted the high-affinity site to a low-affinity one. Thus, 1) the LTD4 receptor is coupled to an IAP-sensitive GTP-binding protein, 2) this GTP-binding protein is dissociable from the receptor by solubilizing the lung membrane with CHAPS and Mg2+, and 3) the receptor associated to or dissociated from a GTP-binding protein exhibited a high- or low-affinity state, respectively. These data provide an insight into the molecular mechanism of regulation of the LTD4 receptor signaling process by association and dissociation with an IAP-sensitive GTP-binding protein.     

Characterization of the cation-binding properties of porcine neurofilaments

In the presence of physiological levels of Na+ (10 mM), K+ (150 mM), and Mg2+ (2 mM), dephosphorylated neurofilaments contained two Ca2+ specific binding sites with Kd = 11 microM per unit consisting of eight low, three middle, and three high molecular subunits, as well as 46 sites with Kd = 620 microM. Only one class of 126 sites with Kd = 740 microM was detected per unit of untreated neurofilaments. A chymotryptic fraction enriched in the alpha-helical domains of neurofilament subunits contained one high-affinity Ca2+-binding site (Kd = 3.6 microM) per domain fragment of approximately 32 kDa. This site may correspond to a region in coil 2b of the alpha-helical domain, which resembles the I-II Ca2+-binding site in intestinal Ca2+-binding protein. Homopolymeric filaments composed of the low or middle molecular weight subunits contained low-affinity Ca2+-binding sites with Kd = 37 microM and 24 microM, respectively, while the Kd values for the low-affinity sites in heteropolymeric filaments were 8-10-fold higher. Competitive binding studies, using the chymotryptic fraction to assay the high-affinity Ca2+-binding sites and 22Na+ to monitor binding to the phosphate-containing low-affinity sites, yielded Kd values for Al3+ of 0.01 microM and 4 microM, respectively. This suggests that the accumulation of Al3+ in neurons may be due in part to its binding to neurofilaments.     

Exploration of the nucleotide binding sites of the isolated ADP/ATP carrier protein from beef heart mitochondria. 1. Probing of the nucleotide sites by Naphthoyl-ATP, a fluorescent nontransportable analogue of ATP

The ADP/ATP carrier protein was extracted and purified from beef heart mitochondria, and its binding parameters with respect to 3'-O-naphthoyladenosine 5'-triphosphate (N-ATP), a fluorescent nontransportable analogue of ATP, were studied. The binding of N-ATP to the isolated carrier protein was accompanied by a decrease in fluorescence. Conversely, the release of bound N-ATP upon addition of carboxyatractyloside (CATR) or ATP resulted in a fluorescence increase. The bound N-ATP that was released upon addition of an excess of CATR or ATP was referred to as specifically bound N-ATP, i.e., N-ATP bound to the nucleotide sites of the carrier protein. Two classes of binding sites for N-ATP could be identified; the number of high-affinity sites (Kd less than 10 nM) was equal to the number of low-affinity sites (Kd = 0.45 microM). CATR behaved apparently as a noncompetitive inhibitor of the binding of N-ATP. The amount of N-ATP released increased linearly with the amount of CATR added, indicating an extremely high affinity of the carrier protein for CATR. The number of CATR binding sites was equal to half the total number of N-ATP binding sites (high- and low-affinity sites); at saturating concentrations of N-ATP, the binding of 1 mol of CATR resulted in the release of 2 mol of bound N-ATP, one from the high-affinity site and the other from the low-affinity site, showing unambiguously that each CATR site is interacting with a pair of probably interdependent N-ATP sites. A clear competition between N-ATP and ATP for binding to the carrier protein was demonstrated. The Kd values of the high- and low-affinity sites for ATP were less than 50 nM and 5 microM, respectively. In the presence of high concentrations of ATP, the two classes of N-ATP binding sites became indistinguishable, suggesting interconversion. It is proposed that the asymmetry in affinity for N-ATP binding is induced by the binding step itself, the carrier protein exhibiting a negative cooperativity for N-ATP binding.     

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process.

The interaction between interleukin IL-1 alpha and PGE2 on P388D1 cells has been investigated. Preincubation of murine macrophage-like cells, P388D1, with IL-1 alpha (0-73 pM) reduced the binding of PGE2 to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1 alpha decreased the PGE2 binding by lowering both the high and low affinity receptor binding capacities (from 0.31 +/- 0.02 to 0.12 +/- 0.01 fmol/10(6) cells for the high affinity receptor binding sites and from 2.41 +/- 0.12 to 1.51 +/- 0.21 fmol/10(6) cells for the low affinity receptor binding sites). However, the dissociation constants of the receptors of the IL-1 alpha-treated cells remained unchanged. Inhibition of PGE2 binding by IL-1 alpha did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1 alpha inhibits the binding of PGE2 to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE2.