EditorialScatchard plots: Common errors in correction and interpretation
References (3)
- D. Rodbard et al.
Cited by (370)
Cooperative binding of annexin A2 to cholesterol- and phosphatidylinositol-4,5-bisphosphate-containing bilayers
2014, Biophysical JournalBiological membranes are organized into dynamic microdomains that serve as sites for signal transduction and membrane trafficking. The formation and expansion of these microdomains are driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Annexin A2 (AnxA2) is a peripherally associated membrane protein that can support microdomain formation in a Ca2+-dependent manner and has been implicated in membrane transport processes. Here, we performed a quantitative analysis of the binding of AnxA2 to solid supported membranes containing the annexin binding lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine in different compositions. We show that the binding is of high specificity and affinity with dissociation constants ranging between 22.1 and 32.2 nM. We also analyzed binding parameters of a heterotetrameric complex of AnxA2 with its S100A10 protein ligand and show that this complex has a higher affinity for the same membranes with Kd values of 12 to 16.4 nM. Interestingly, binding of the monomeric AnxA2 and the AnxA2-S100A10 complex are characterized by positive cooperativity. This cooperative binding is mediated by the conserved C-terminal annexin core domain of the protein and requires the presence of cholesterol. Together our results reveal for the first time, to our knowledge, that AnxA2 and its derivatives bind cooperatively to membranes containing cholesterol, phosphatidylserine, and/or phosphatidylinositol-4,5-bisphosphate, thus providing a mechanistic model for the lipid clustering activity of AnxA2.
Androgen receptor in the oviduct of the turtle, Trachemys scripta
2005, Comparative Biochemistry and Physiology - B Biochemistry and Molecular BiologyCirculating androgens reach high concentrations in females of some reptiles and amphibians. We are testing the hypothesis that androgens can act directly in female reptilian reproductive tissues, via the androgen receptor. In this study, we sought to determine if androgen receptors are present in the oviduct of the turtle, Trachemys scripta, using radioligand-binding assays and immunological assays. An androgen-binding site was detected in turtle oviductal cytosol and oviductal nuclear extract by radioligand binding assay, using 3H-dihydrotestosterone (DHT) as the ligand. This site was saturable (Bmax=11 pmol/g tissue), had a high affinity (10−10 M), and showed specificity typical of androgen receptors (DHT>testosterone, progesterone≫estradiol, cortisol). Western blotting using an anti-androgen receptor antibody revealed a band of immunoreactivity in oviductal cytosol at approximately 115 kDa, and a more prominent band at 50 kDa, possibly indicating a truncated form of the androgen receptor. Immunohistochemistry revealed crossreactivity of the androgen receptor antibody against oviductal glandular cells but not against oviductal luminal epithelial or muscularis cells. The presence of androgen receptor in the turtle oviduct suggests that androgens have a role in female reproduction and that their action can be mediated directly by androgen receptor.
Requirements for reliable determination of binding affinity constants by saturation analysis approach
2004, Journal of Steroid Biochemistry and Molecular BiologyAccurate calculation of the equilibrium association constant (K) and binding site concentration (N) related to a receptor (R)/ligand (L) interaction, via R saturation analysis, requires exact determination of the specifically bound L concentration (BS) and the unbound L concentration (U) at equilibrium. However, most binding determinations involve a procedure for separation of bound and unbound L. In such situations, it was previously shown that correct calculation of BS and U from binding data requires prior determination of α, i.e. the procedure parameter representing the proportion of equilibrium BS recovered after running the separation process, and of kn, i.e. the equilibrium nonspecific binding coefficient. For the simplest model of R/L interaction, the consequences of α neglect and/or kn neglect on determination of K and N, via R saturation analysis, are investigated. When α but not kn has been determined, BS can be accurately calculated, whereas U is overestimated by factor (kn + 1). Consequently the type (linear or hyperbolic) of theoretic curves obtained by usual representations (such as the Scatchard, the Lineweaver–Burk or the Michaelis–Menten plot) of the R/L binding is unchanged; these curves afford correct N and underestimation of K by factor (kn + 1). When α (α < 1) has not been determined BS and U are underestimated and overestimated, respectively. Then erroneous representations of the R/L binding result (e.g. instead of regular straight line segments, Scatchard plot and Lineweaver–Burk plot involve convex-upward and convex-downward hyperbola portions, respectively, suggestive of positive cooperativity of L binding), which leads to incorrect N and K. Errors in N and K would depend on (i) the binding (K, N and kn) and method (α) parameters and (ii) the expressions used to calculate approximate BS and U values. Simulations involving variable α, KN and kn values indicate that: (1) the magnitude of error in N determination (mainly involving moderate underestimation) directly depends on the α value; (2) the magnitude of K underestimation mainly depends on the KN value; it is moderate (usually < two-fold) with KN values < 1, but could become very high (e.g. >100-fold), when KN > 102. In this case, the K underestimation is modulated by the α and kn values. Practical situations which afford high KN and thus might result in very marked underestimation of K are discussed. A single R dilution method is proposed to assess the validity of K determinations using the R saturation analysis approach.
Characterization of [<sup>3</sup>H]flunitrazepam binding to melanin
2001, Analytical BiochemistryIn both clinical and forensic toxicology, the analysis of hair for drugs is an important tool to determine drug use in the past or to verify abstinence from illegal drugs during extended periods. Melanin is proposed as one of the factors that influences drug incorporation to hair and we have characterized the binding of the drug flunitrazepam to melanin in vitro. The drug was 3H labeled and melanin granules from cuttlefish, Sepia officinalis, were used according to the suggested standard for melanin studies. We observed a rapid Langmuir-like binding followed by a slower diffusion-limited binding that may be interpreted as an initial surface binding followed by deeper bulk binding. From three concentrations of melanin, with a 60-min incubation time, a mean saturation value of 180 ± 20 pmol/mg was calculated. The binding of a group of benzodiazepines and tranquilizers was compared to the binding of [3H]flunitrazepam by means of displacement experiments. These drugs showed binding characteristics similar to [3H]flunitrazepam except phenobarbital, which had a lower affinity to melanin. The method presented in this study allowed measurements with low melanin and drug concentrations and it has the strength of directly measuring the amount of drug bound to melanin, in contrast to previous indirect methods.
Characterization of subclones of the epithelial cell line from Chironomus tentans resistant to the insecticide RH 5992, a non-steroidal moulting hormone agonist
2000, Insect Biochemistry and Molecular BiologySelection of hormone resistant subclones in the continuous presence of the insecticide and ecdysteroid mimick RH 5992 (tefubenozide) resulted preferentially in clones with defects in ecdysteroid receptor function. RH 5992 is already degraded to polar products in wild-type cells; no increase in metabolism of tefubenozide is observed in resistant clones. According to Western blots, ecdysteroid receptor (EcR) and its heterodimerization partner ultraspiracle (USP) are present in all resistant clones. The concentrations are comparable to wild-type cells, but in three clones the extent of phosphorylation of USP is diminished. With regard to hormone binding several types of hormone resistance are distinguished: (1) The same two high-affinity hormone recognition sites are present as in wild-type cells (KD1=0.31±0.28 nM, KD2=6.5±2.4 nM) but the number of binding sites is reduced. (2) The binding site with the lower affinity (KD2) is missing. (3) The binding site with the higher affinity (KD1) is missing. (4) No specific binding is observed. Ponasterone A binding can be rescued by addition of EcR but not by USP. (5) Ligand specificity is altered. RH 5992 can not compete [3H]-ponasterone A as efficient as in wild-type cells.
Nicotine binding to native and substituted peptides comprising residues 188-207 of nicotinic acetylcholine receptor α1, α2, α3, α4, α5, and α7 subunits
2000, Biochemical and Biophysical Research CommunicationsStructural determinants of L-[3H]nicotine binding to synthetic peptides comprising residues 188–207 of nicotinic acetylcholine receptor α subunits were invesitigated by equilibrium binding analysis. Two binding components were detected, one of low affinity (Kd ∼ 1.5 μM) that did not differ significantly among peptides and another of high affinity. The high affinity binding component was higher for the neuronal peptides (Kd = 14–23 nM) than the muscle α1 peptides (Kd = 52 nM). The following nonconservative substitutions in the α4 peptide resulted in a significant decrease in nicotine affinity for the peptide: Y190A, Y190D, C192G, E195A, E195–, P199A, P199–, and Y203A. Substitution of α4P199 with a leucine which is present in the α1 sequence decreased the affinity of the α4 peptide for nicotine and substitution of α1L199 with a proline (α4) or a glutamine (α3) increased the affinity of the α1 peptide. It is concluded that aromatic residues contribute to the binding site for nicotine on the α4 subunit and that the residue present at position 199 partly determines differences in nicotine affinity for different α subunits.