Cytokinin-binding proteins

This article is focused on the modalities of reception of cytokinins which remain largely unknown. It summarizes the main steps of the different protocols used to study cytokinin-binding proteins (CBPs). We place emphasis on the significance and specificity of the detection according to the properties of the probes used: radioactive or photoreactive cytokinins, fluorescent anticytokinins, anti-idiotype antibodies. The purification procedures are also examined. The cellular localisation and the putative physiological roles of the numerous and different CBPs found are considered. The interest of genetic and molecular studies is discussed.     

Generalized binding phenomena in an allosteric macromolecule

A general macromolecular partition function is developed in terms of chemical ligand activity, temperature and pressure for systems described by an array of species which are characterized by their state of allosteric conformation and ligand stoichiometry. The effects of chemical ligand binding, enthalpy change, and volume change are treated in a parallel manner. From a broad viewpoint all of these effects can be regarded as specific cases of generalized binding phenomena. This approach provides a general method for analyzing calorimetric and ligand binding experiments. Several applications are given: (1) Thermal scanning data for tRNAphe (P.L. Privalov and V.V. Filimonov, J. Mol. Biol. 122 (1978) 447) are shown to fit a general model with six conformational states. By application of linkage theory it is shown that sodium chloride is expelled as the molecule denatures. (2) The results of calorimetric titrations on the arabinose binding protein (H. Fukada, J.M. Sturtevant and F.A. Quiocho, J. Mol. Biol. 258 (1983) 13193) are shown to fit a simple two-state allosteric model. (3) A thermal binding curve is simulated for an unusual respiratory protein, trout I hemoglobin (B.G. Barisas and S.J. Gill, Biophys. Chem. 9 (1979) 235), in order to illustrate both the similarities and differences between enthalpy and chemical ligand binding processes.     

Depolarization-Induced Increase in Surface Binding and Internalization of 125I-Nerve Growth Factor by PC 12 Pheochromocytoma Cells

The binding and internalization of 125I-nerve growth factor (NGF) by PC12 pheochromocytoma cells was studied as a function of extracellular potassium concentration. Both surface-bound and internalized fractions of 125I-NGF associated with the cells under depolarizing conditions (50 mM K+) increased to 144 +/- 28% (average +/- SEM, six different cell preparations) and to 176 +/- 12% (n = 6), respectively, of those observed at 6.0 mM K+. Scatchard-type analysis of the data indicates increased sites for the binding and internalization of iodinated NGF by the cells. Similar enhancement was observed for cells treated with NGF as well. This voltage-dependent phenomenon was reversible, and also observed in the presence of veratridine. Moreover, withdrawal of extracellular Ca2+ abolished high K+-induced modulation of 125I-NGF binding and internalization, indicating that this effect may be mediated by Ca2+.     

Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

At 0°C, when Na⁺ was the only cation present in the incubation medium, increasing the Na⁺ concentration from 3 to 10 mM enhanced the affinity of [³H]l-[2-(di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([³H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5_max. For higher Na⁺ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na⁺. In a 10 mM Na⁺ medium, the K_D and the B_max were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [³H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na⁺. A similar Na⁺ dependence was observed at 20°C. Scatchard plots indicated that K⁺, Ca²⁺, Mg²⁺, and Tris⁺ acted like competitive inhibitors of the specific binding of [³H]GBR 12783. The inhibitory potency of various cations (K⁺, Ca²⁺, Mg²⁺, Tris⁺, Li⁺ and choline) was enhanced when the Na⁺ concentration was decreased from 130 to 10 mM. In a 10 mM Na⁺ medium, the rank order of inhibitory potency was Ca²⁺ (0.13 mM) > Mg²⁺ > Tris⁺ > K⁺ (15 mM). The requirement for Na⁺ was rather specific, because none of the other cations acted as a substitute for Na⁺. No anionic requirement was found: Cl^-, Br^-, and F^- were equipotent. These results suggest that low Na⁺ concentrations are required for maximal binding; higher Na⁺ concentrations protect the specific binding site against the inhibitory effect of other cations.     

Solubilization and Characterization of Calcitonin Gene-Related Peptide Binding Site from Porcine Spinal Cord

The binding site for calcitonin gene-related peptide (CGRP) was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in an active form from porcine spinal cord. 125I-labeled human alpha-CGRP (125I-CGRP) binding to the solubilized protein was determined by filtration using a GF/B glass filter. The maximal binding activity (approximately 60% of the crude membrane fraction) was obtained with 5 mM CHAPS. 125I-CGRP binding to the solubilized protein was of high affinity, saturability, and high specificity, having KD and Bmax values of 3.69 pM and 338 fmol/mg of protein, respectively. The binding activity was eluted in a single peak with a molecular mass of 400,000 daltons by gel filtration on TSK gel G4000SW. These results suggest that the solubilized protein may be responsible for the specific binding site.     

小麦叶绿体中CTK结合蛋白的结合活性研究

叶绿体中存在着与细胞分裂素(CTK)专一结合的蛋白质。这一蛋白与6-苄氨基嘌呤(6 BA)的亲和力很强,解离常数达3.7×10~(-8)mol/L。最大结合量为10.7 pmol 6 BA/mg蛋白,Seatchard分析表明只有一类结合位点。不同的叶绿体纯化步骤对CTK结合蛋白的活性有不同的影响,分离步骤少而快速的差速离心法可以得到具有较高结合活性的叶绿体。叶绿体经分离纯化后,低温保存时的结合活性较稳定,-20℃以下可以较长期保存。用EDTA预处理叶绿体,不降低CTK结合蛋白对6 BA的结合活性,而用高浓度的NaCl处理,可以使叶绿体结合6BA的能力明显下降。这说明EDTA不能使CTK结合蛋白从叶绿体膜系统表面解离,而高浓度NaCl则有这种可能性。     

Quantitative autoradiographic analysis of [I]-human CGRP binding sites in the dorsal horn of rat following chronic constriction injury or dorsal rhizotomy

Quantitative receptor autoradiography was used to examine the binding of [¹²⁵I]-human CGRP in the dorsal horn of the L4 spinal segment of rats with a chronic constriction injury (CCI) of the sciatic nerve or unilateral dorsal rhizotomies of spinal segments L1–L6. At the times selected for study, we found no change in the amount of CGRP binding in any areas examined following CCI. In contrast, our results showed a temporally related increase in the amount of CGRP binding in areas within laminae I–II and in lateral lamina V of the dorsal horn ipsilateral to the rhizotomies. These results indicate that CGRP binding sites are regulated, most likely, by changes in the release of CGRP. Further, our results suggest that the release of CGRP from primary afferent neurons is unchanged in animals with a CCI.     

Specific Binding of Glycosylated β-Galactosidase to Bovine Brain Synaptic Membrane

The binding of a series of glycosylated beta-galactosidases to a fraction rich in synaptic membrane of bovine brain was examined. beta-galactosidase modified with p-aminophenyl beta-D-galactopyranoside (beta-D-Gal beta-gal) was found the most effective in binding to synaptic membrane, followed by that modified with beta-D-glucopyranoside, whereas the enzyme modified with p-aminophenyl derivatives of alpha-D-galactopyranoside, alpha-D-glucopyranoside, and alpha- and beta-L-fucopyranoside were found not to bind to the membrane. The binding was dependent on time, temperature, and pH; the maximal binding was obtained within 15 min at 4 degrees C and the optimal pH was approximately 4.0. The binding of beta-D-Gal beta-gal was inhibited by free p-aminophenyl beta-D-galactopyranoside and by the treatment of synaptic membrane with trypsin or phospholipase A2 or C. The equilibrium dissociation constant and the maximal concentration of binding sites were determined by Scatchard analysis to be 470 +/- 35 nM and 27.5 +/- 3.1 pmol/mg protein (n = 1). The results suggest that a specific binding site for the specified carbohydrates exists in synaptic membrane and is involved in the internalization of glycoconjugates into nerve terminals.     

Postmortem Changes in Binding to the Muscarinic Receptor from Human Cerebral Cortex

Abstract: The effects of storage at 4°C on the antagonist and agonist binding properties of the muscarinic acetyl-choline receptor from fresh surgical and frozen autopsy samples from human cerebral cortex were studied. The number of 1-[³H]3-quinuclidinyl benzilate binding sites and their affinities were stable up to 51 h, both when stored as pieces of intact nonfrozen tissue and as a homogenate. The agonist binding properties as measured by the ability of the muscarinic agonist carbachol to compete with l-[³H]3-quinuclidinyl benzilate were also stable up to 51 h when the tissue was stored in the form of pieces. The affinity for carbachol decreased when the tissue was stored as a homogenate. The frozen autopsy samples showed no significant differences in binding properties in comparison with fresh neurosurgical tissue.     

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [3H]Flunitrazepam and [3H]Ethyl-β-carboline-3-carboxylate Binding Sites in Rat Brain Membranes

Abstract: This study was designed to analyze possible differences in the binding of [³H]flunitrazepam ([³H]FNZP) and [³H]ethyl - β - carboline - 3 - carboxylate ([³H]β-CCE), to rat brain membranes, in various experimental conditions. In cerebral cortex, hippocampus, cerebellum, and orain stem the number of binding sites for [³H]β-CCE was higher than for [³H]FNZP; both were displaced by clonazepam. Until the 7th day of postnatal brain development the numbers of [³H]FNZP and [³H]β-CCE sites were equivalent; but later on, the β-carboline sites increased to a higher level. Noradrenergic denervation by 6-hydroxydopamine was followed in the hippocampal formation. Already after 2 days, there was a decrease in [³H]FNZP sites, which reached 70% of control after 14 days. Similar results were obtained with DSP-4 denervation. This change was only in B_max and not in K_D, In contrast, the [³H]β-CCE sites did not change with denervation. Neonatal injection of l - 2,4,5 - trihydroxyphenylalamine or DSP-4 produced in the adult a decrease in [³H]FNZP sites in the cerebral cortex, in parallel with the noradrenergic denervation. On the other hand, there was an increase in the cerebellum and brain stem, in correspondence with the hyperinnervation by sprouting. In these rats, the number of sites for [³H]β-CCE did not change in the different brain regions. With 0.1% Triton X-100, applied to synaptosomal membranes, [³H]FNZP binding was reduced by 35%, while that of [³H]β-CCE was not significantly changed. These results suggest that there is heterogeneity of binding sites for benzodiazepine receptors in rat brain. A tentative interpretation of the experiments involving noradrenergic denervation and hyperinnervation, as well as those with Triton X-100, is that [³H]FNZP binds to pre- and postsynaptic receptors, while [³H]β-CCE binds mainly to postsynaptic benzodiazepine receptors.