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Spectrophotometrically monitored ligand titration is an important method for the determination of equilibrium dissociation constants (K(d)) from nitric oxide synthases (NOS). Low K(d) sites such as the tetrahydrobiopterin and arginine binding sites present difficulties in that experiments often require enzyme concentrations of the same magnitude as the K(d). An analytical method based on computer simulation is described that allows the estimation of K(d) values without an independent means of monitoring free ligand or without an accurate prior determination of the number of binding sites. The K(d) for arginine is approximately 0.5 microM for the tetrahydrobiopterin replete neuronal and inducible isoforms (nNOS and iNOS), while the endothelial isoform has a slightly higher K(d) (1.5 microM). N-OH-arginine (an intermediate) binds to nNOS with a K(d) of around 0.2 microM, while the inhibitors N-methyl-arginine and N-nitro-arginine bind more tightly; our best K(d) estimates are 100 nM or lower.
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Organic probe molecules have recently been used to define hydrophobic binding sites on the surface of proteins. It appears that the presence of water on the surface of a protein plays a crucial role in the interaction between that protein and its binding site.
Binding sites for the dipeptide L-carnosine (beta-alanyl-L-histidine) have been detected in membranes prepared from mouse olfactory bulbs. The binding of L-[3H]-carnosine was saturable, reversible and stereospecific and had a Kd of about 770 nM. The stereospecific binding of L-carnosine represented about 30% of the total binding at pH 6.8, and decreased markedly with increasing pH. Binding was stimulated by calcium, unaffected by zinc, magnesium or manganese and inhibited by sodium and potassium. Carnosine binding was sensitive to trypsin and phospholipases A and C, but not to neuraminidase. Nystatin and filipin, which interact with membrane lipids, also interferred with binding. Some peptide analogues of carnosine were potent inhibitors of binding, but a variety of drugs serving as potent inhibitors in other binding systems had no effect on carnosine binding. Carnosine binding to mouse olfactory bulb membranes was 15-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than in cerebellum membranes and 3-fold higher than in membranes from spinal medulla and the olfactory tubercle-lateral olfactory tract area. Binding sites for 6 other radiolabeled receptor ligands were also detected in bulb membranes. Peripheral deafferentation of the olfactory bulbs by intranasal irrigation with ZnSO4 led to a loss greater than 90% of the L-[3H]carnosine binding in 4--5 days with much smaller losses in binding of the other 6 ligands over a 180-day observation period. This initial loss of carnosine binding after denervation was due to a loss of binding site stereo-specificity followed by a loss of binding sites. The characteristics of the carnosine binding site in olfactory bulb fulfil 6 of the 7 criteria considered relevant for a functional receptor.
Binding characteristics of two types of ligands for human neo-C-reactive protein (neo-CRP), which is a conformationally altered but physiologically relevant form of CRP, were studied fluorometrically by probing CRP immobilized on a polystyrene surface with europium-labeled ligands. Two Eu-ligands used were bovine serum albumin derivatives that contain on average 40 residues of ligand structures, one derivative containing phosphorylcholine (PC) and the other lactosyl residues. The PC-containing ligands required the presence of calcium for binding, whereas galactose-containing derivatives bound in the absence of calcium. The optimal pH for the PC-dependent binding was broad (pH 6-8), whereas the best binding pH for the galactose-dependent binding was around 6. The carbohydrate-mediated binding is rather nonspecific: the binding site prefers galactose configuration, but other hexoses can be accommodated. The two best monosaccharide inhibitors at this site were galactose-6-phosphate and galacturonic acid, suggesting the importance of having a negatively charged group at C-6 position of galactose. In fact, the phosphate-binding site is common to both PC and sugar phosphates, and the choline- and the sugar-binding sites are probably located on either side of the phosphate-binding site. Binding characteristics of Eu-labeled PC-BSA to neo-CRP are quite similar to that found for native CRP in solution phase [Lee et al. (2002) J. Biol. Chem., 277, 225-232], whereas binding of sugar phosphates by neo-CRP shows considerably less stringent requirements compared to native CRP. For instance, galactose-alpha1-phosphate was not inhibitory at all in the native CRP binding assay, whereas it was a good inhibitor in the neo-CRP assay.
Binding of the alpha-adrenergic antagonists [3H]prazosin and [3H]rauwolscine to well-characterized subcellular membrane fractions isolated from dog mesenteric arteries and veins was studied. Binding of both ligands was saturable with Kd values of 0.5 +/- 0.1 nM for [3H]prazosin and 5.85 +/- 0.85 nM for [3H]rauwolscine in arteries, and 0.87 +/- 0.4 nM for [3H]prazosin and 6.6 +/- 1.5 nM for [3H]rauwolscine in veins. In veins, the maximum number of binding sites for [3H]rauwolscine was higher than that for [3H]prazosin, whereas in arteries the maximum number of binding sites for each ligand was similar. In microsomes from dog aorta, the maximum number of bindings sites for [3H]prazosin was higher than that for [3H]rauwolscine. Neuronal membrane contamination in these studies was minimized by dissection procedures and evaluated by the comparison of [3H]saxitoxin binding in various preparations. Only mesenteric veins responded functionally to agonists acting on alpha 2 adrenoceptors. This study thus identified two distinct populations of [3H]prazosin and [3H]rauwolscine binding sites in the plasma membranes of dog mesenteric vessels and suggests that a much higher density of alpha 2-compared to alpha 1-adrenoceptor binding sites is required for a contractile response.
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Binding sites having the characteristics of receptors for "activated" alpha 2-macroglobulin (alpha 2M) have been solubilized with octyl-beta-D-glucoside from fibroblast membranes. When the detergent was removed by dialysis, the resulting insoluble extract was shown to bind 125I-alpha 2M specifically. Analysis of the binding data using a nonlinear curve-fitting program suggests that the solubilized preparation contains two classes of binding sites (KD = 0.34 nM and KD = 104 nM). Membranes or solubilized extracts from KB cells which lack alpha 2M binding sites did not specifically bind 125I-alpha 2M. The solubilized binding sites from fibroblasts were inactivated by boiling and trypsin treatment, and required Ca+2 for maximal binding. In addition, the high affinity binding of 125I-alpha 2M to the solubilized receptor was inhibited by bacitracin and by alpha-bromo-5-iodo-4-hydroxy-3-nitroacetophenone, two agents which interfere with the uptake of alpha 2M in cultured fibroblasts. Using a combination of ion exchange and gel permeation chromatography, we have purified the high affinity alpha 2M binding site approximately 100-fold from membrane derived from NIH-3T3 (spontaneously transformed) fibroblasts grown as tumors in mice. The receptor is apparently an acidic protein and the receptor octyl-beta-D-glucoside complex has a Stokes radius of 45-50 A as measured by gel filtration.
Binding and competition among Cry1Aa, Cry1Ac, and Cry1Ba toxins were analyzed quantitatively in vitro by using (sup125)I-labeled activated toxins and brush border membrane vesicles isolated from Chilo suppressalis larval midguts. The three toxins bound specifically to the midgut brush border membrane vesicles. Direct binding experiments showed that Cry1Aa and Cry1Ba recognized a single class of binding sites with different affinities, whereas Cry1Aa recognized two classes of binding sites, one with a high affinity and a low concentration and the other with a lower affinity but higher concentration. Competition experiments showed that toxins Cry1Ac and Cry1Ba shared a binding site in the C. suppressalis midgut membranes and that this site was also the low-affinity binding site for Cry1Aa.
Binding of basic fibroblast growth factor (FGF2) to its high affinity receptors requires the presence of specific heparan sulfate (HS) moieties on the cell surface that act as coreceptors. To determine the contribution of cell-surface HS to modulation of FGF2-dependent cell growth, we studied the changes in the cell mass and FGF2 binding of endothelial cell HS under normoxic and hypoxic conditions in vitro. Both large vein and cardiac microvascular endothelial cells cultured under hypoxic conditions demonstrated an increase in the ratio of cell-surface HS to chondroitin sulfate (CS), as well as an increase in the number of low affinity (HS-associated) binding sites for FGF2 with no change in the apparent K(d). This increase in the number of HS-FGF2 binding sites, in the absence of a significant change in FGF receptor expression, resulted in enhanced responsiveness of hypoxic, compared with normoxic, endothelial cells to FGF2 stimulation. Gene expression studies demonstrated increased expression of the key regulatory enzyme responsible for HS chain synthesis, 1,4 GlcNAc transferase (GlcNAcT-I), as well as increased expression of 2-O sulfotransferase (HS2ST), the enzyme responsible for sulfation of IdoA, a crucial part of the HS-FGF2 binding site. Transduction of cells with an adenovirus encoding a HIF-1alpha expression construct resulted in a similar increase in GlcNAcT-I and HS2ST expression. We conclude that hypoxia increases endothelial cell responsiveness to FGF2 by promoting preferential synthesis of HS rather than CS chains and increasing the number of FGF2-binding sites on HS chains. Both of these events are mediated by a HIF-1alpha-dependent increase in expression of the enzymes GlnNAcT-I and HS2ST. This shift in cell-surface HS composition results in enhanced cell sensitivity to FGF2-induced growth stimulation.
A new method for mapping RNA initiation, termination, and splice sites was developed. The method involves: 1) hybridization of RNA to end-labeled single-stranded DNA; 2) mild digestion of the hybrid with a single strand specific nuclease; 3) high resolution gel electrophoresis and autoradiography. The regions of the labeled probe which are resistant to nuclease digestion are mapped by measuring the distance from the labeled end. The sequence can be determined by running the end-labeled probe sequenced according to the protocol of Maxam and Gilbert (Maxam, A.M., and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) at the same time. The feasibility of this method was tested with adult chicken beta-globin mRNA, and we found that the transcription initiation, termination, and RNA splice sites can be determined to within a few bases of the known sites. Using this method we also found that ribosomes bind to beta-globin mRNA from 22 bases upstream from the translation start codon (AUG) to 15 bases past the stop codon (UAA).
Binding of 125I-labeled rat (r) PTH-(1-34) to ROS 17/2.8 osteoblastic bone cells and to membranes from these cells was examined. Competitive binding inhibition experiments were performed using unlabeled rPTH-(1-34) with particular emphasis on concentrations of peptide below 1 nM. In intact cells, binding of labeled rPTH-(1-34) was highly specific, and inhibition of binding by unlabeled ligand suggested the presence of two classes of binding sites, one with high affinity and low capacity (KD = 40 pM, approximately 20% of total binding sites) and the other with lower affinity and high capacity (KD = 2 nM, approximately 80% of the sites). Membranes prepared from ROS cells also exhibited a pattern of binding from competitive inhibition curves consistent with two distinct binding sites (KD = 30 pM and 6 nM). In intact ROS cells, cellular cAMP levels increased over the range of 10(-11)-10(-9) M rPTH-(1-34) with an ED50 intermediate between the two KD values (0.25 nM). These data suggest that osteoblastic bone cells possess two distinct classes of membrane receptors for PTH. Since the KD of the higher affinity site more closely approximates circulating concentrations of PTH, binding to this site may have physiologic relevance.
Binding at the mu, delta- and kappa-types of opioid binding sites was compared in homogenates from the brains of guinea-pig, rabbit, rat and two mouse strains, under conditions of selective labelling. Species differences were shown by two observations. Firstly, analysis of saturation curves in homogenates of brain from which the cerebellum had been removed showed that in guinea-pig brain the opioid binding sites consist of 24% mu-sites, 32% delta-sites and 44% kappa-sites. In contrast, in rabbit brain the corresponding values are 43% mu-sites, 19% delta-sites and 37% kappa-sites and in rat brain, 46% mu-sites, 42% delta-sites and 12% kappa-sites. In the brains of DBA/2 mice the opioid binding sites are comprised of 51% mu-sites, 29% delta-sites and 20% kappa-sites and in C57BL/10 mice, of 44% mu-sites, 35% delta-sites and 21% kappa-sites; these strain differences are due to significant differences in the concentrations of the mu-sites. Secondly, species differences were found when the binding of single concentrations of tritiated ligands (1 X KD value in whole brain) was determined at mu-, delta- and kappa-sites in six brain regions from guinea-pig, rat or rabbit.
Binding of the BZLF1 viral transactivator to Epstein-Barr virus (EBV) oriLyt has been reported to be essential for viral DNA replication. We have constructed a recombinant virus (E2-oriLyt-R) in which the oriLyt BZLF1-binding sites (ZRE) were exchanged against papilloma E2-binding sites. A fusion protein between the BZLF1 protein-transactivating domain and the E2 protein-binding domain was able to reactivate lytic replication in E2-oriLyt-R. However, BZLF1 alone could also induce E2-oriLyt-R, albeit with much lower efficiency. ZRE are therefore important but not absolutely essential cis elements for lytic replication. This shows the importance of recombinants to evaluate viral functions.
Binding sites for one Pi and two ATP or ADP molecules have been identified on the isolated, reconstitutively active beta subunit from the Rhodospirillum rubrum F0.F1 ATP synthase. Chemical modification of this beta subunit by the histidine reagent diethyl pyrocarbonate or by the carboxyl group reagent Woodword's reagent K results in complete inhibition of Pi binding to beta. The same reagents inhibit the binding of ATP to a Mg-dependent low-affinity site but not to a Mg-independent high-affinity site on this beta subunit. The binding stoichiometry of ADP to either site is not affected by these modifications. The beta subunit modified by either one of these reagents retains its capacity to rebind to beta-less chromatophores but not its ability to restore their photo-phosphorylation. These results indicate that the low-affinity Pi binding site on beta is located at the binding site of the gamma-phosphate group of ATP in the Mg-dependent low-affinity nucleotide binding site. This site contains histidine and carboxyl group residues, both of which are required for the binding of Pi and of the gamma-phosphate group of ATP. The same residues must also be involved in the capacity of the isolated beta subunit to restore the catalytic activity of the beta-less ATP synthase. It is therefore concluded that the low-affinity Mg-dependent substrate binding site identified on the isolated beta subunit of the R. rubrum F0.F1 ATP synthase is the catalytic site of this enzyme complex.
Binding of lambda phage cro repressor to the synthetic half of OR3, the most conservative half of the specific binding sites, was investigated by proton nuclear magnetic resonance spectroscopy. It was found that the alpha-helical segment (27-36) of the protein was involved in specific interactions with the model binding site. The 3-dimensional structure of cro repressor does not change noticeably upon complex formation. Intercalation can be excluded as a possible means of interaction.
The mechanisms for regulating platelet HDL3 binding sites were investigated. HDL3 binding was rapid (T(1/2) association=4 minutes) and completely reversible (T(1/2) dissociation=14.5 minutes) at 4 degrees C, 22 degrees C, and 37 degrees C, and kinetic analysis yielded forward and reverse constants of 7.3x10(-4) x s(-1) and 7.13x10(3) x s(-1) x M(-1), respectively. Nevertheless, neither inhibitors of binding sites recycling or of pinocytosis, such as ammonium chloride, chloroquine, monensin, colchicine, and sodium azide, modified the binding characteristics. Moreover, when platelets were loaded with cholesterol, binding sites were not regulated (up or down). However, when exposed to high concentrations of HDL3 (1.5 g/L), apoE-free HDL (1.5 g/L), HDL2 (0.5 g/L), apoE-rich HDL (0.5 g/L), and VLDL (0.3 g/L) there was rapid downregulation of the number of binding sites in isolated permeabilized platelets, as shown by the reduction of Bmax to 66%, 58%, 45%, 53%, and 51%, respectively. Downregulation was rapid, reversible, and dose and time dependent. In contrast, LDL (up to 2.0 g/L), IDL (up to 0.1 g/L), and chylomicrons (up to 0.5 g/L) had no effect. Protein kinase C inhibitors (150 nmol/L staurosporine, 100 micromol/L H-7, and 10 nmol/L bisindolylmaleimide) inhibited downregulation up to 62% (as average value). The role of the PKC activation in regulating the activity of HDL3 binding sites also was analyzed by determining the cytosol-to-membrane translocation of enzymatic activity. Downregulation mediated by HDL3 rapidly translocated PKC activity (21% +/- 11 of total PKC activity was membrane-associated in control platelets vs. 55+/-8% in downregulated platelets, mean+/-SEM, n=3). However, agents that block sequestration (0.30 g/L, concanavalin A), and other protein kinase inhibitors, such a cAMP-dependent protein kinase inhibitors (1 micromol/L, PKI), and beta2-adrenergic receptor kinase inhibitors (100 nmol/L, heparin) had no effect. The results show that neither endocytotic response nor cholesterol-dependent mechanisms participate in the modulation of platelet HDL3 binding sites. However, a new regulatory mechanism that involves PKC-dependent downregulation of the number of binding sites may be an important pathway to regulate the thrombogenicity of lipoproteins and their effects on platelet reactivity.