Binding stoichiometry of a fluorescent cGMP analogue to membranes of retinal rod outer segments

Abstract

The high-affinity binding of the cGMP analogue 8-(5-thioacetamidofluorescein)-cGMP to rod outer segment membranes depleted of peripherally bound proteins has been defined by equilibrium dialysis (mean SD) : (a) membranes contain about one cGMP binding site per 130 rhodopsin molecules; (b) the concentration of free ligand for half saturation is 2.0 f 0.6 pM; (c) the apparent Hill coefficient of the bound versus free ligand relationship is 1.7 f 0.5; (d) half saturation of the binding sites is sufficient for 85% activation of calcium permeability. A gating mechanism is proposed.The first demonstration of a cation permeability activated directly by cGMP in ROS membranes raised the possibility that cGMP might keep the plasma membrane of the rods permeable in darkness and that in light its hydrolysis might reduce the cell permeability [l]. The former hypothesis has been demonstrated recently [2] while the latter, which has been extensively discussed [3 -61, receives more credit now because of the convergence of in vitro and electrophysiological observations [7-101. A cation permeability activated directly by cGMP in membranes depleted of peripherally bound proteins obviously implies binding of cGMP to the membranes, but this binding has not been shown so far for two reasons: (a) the kinetics of binding and dissociation is likely to be very fast, and this precludes any method based on the physical separation of bound from free ligand; (b) the binding capacity is small and the apparent affinity for the effect relatively low [I -71. While the first problem has been solved working at binding equilibrium, the second has been overcome by working with high concentrations of membranes and using a new fluorescent analogue of cGMP with high affinity to ROS membranes.A study of the activity of this and other analogues of cGMP will precede the binding experiments and the results will be compared to the binding data. MATERIALS AND METHODS Materials -(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-iodoacet-arnido-benzoic acid.[3H]inulin from New England Nuclear; reagents and apparatus for electrophoresis from Bio-Rad; DEAE-cellulose from Whatman; all other reagents from Carlo Erba. Dimethylformamide was redistilled under reduced pressure and methanol was redistilled anhydrous. Synthesis of cGMP analoguesThe Et3N salt of 8-bromo-cGMP was prepared as follows. 200 mg cGMP (Na salt) dissolved in 7 ml 1 M sodium acetate/ acetic acid buffer at pH 3.9 and 3.5 ml bromine water (bromine/water, ljl00, v/v) were left to react in room light for 5 h. The excess of bromine was purged from the solution with air and the product was isolated by DEAE-cellulose (DE 52) ion-exchange chromatography with an Et3N bicarbonate gradient according to Haley [l ll.The Et3N salt of 8-azido-cGMP was prepared by reacting 8-bromo-cGMP with Et3N azide in dry dimethylformamide/ isobutyric acid strictly as described by Haley [I I].It was checked that the product was photolysable. The Et,N salt of 8-thiobenzyl-cGMP [I21 was prepared by refluxing 0.45 g thiourea and 1.35 g 8-bro...

cGMP-dependent channel protein from photoreceptor membranes: single-channel activity of the purified and reconstituted protein.

“…2A, the EC50 = 30 ,uM and the cooperativity n = 2.1. Results from five experiments gave EC50 = 31 + 15 ,M (mean ± SD) and n = 2.3 + 0.5-i.e., similar to the EC50 constant and the cooperativity n of the activation of the channel in situ in excised patches of the plasma (1-6) and disk membrane (12)(13)(14). Fig.…”

ATP can promote activation and deactivation of the rod cGMP‐phosphodiesterase

“…The role of cGMP and ATP as cofactors in the enzyme cascade of the rod has been the subject of various investigations: cGMP is a messenger of the amplified light signal from the R/G/PDE cascade to the light-dependent plasma membrane conductances [3]; in addition, cGMP has been shown to control a disc membrane conductance [4,5]. In frog ROS, cGMP-dependent phosphorylation has been described for two small peptides, components I and II [6] and an effect of cGMP on the binding of GTP and GDP to the G-protein has been demonstrated recently [7].…”

Novel N7- and N1-Substituted cGMP Derivatives Are Potent Activators of Cyclic Nucleotide-Gated Channels

“…While vinyl sulfones are highly selective for reaction with thiols, given elevated pH (>9) and long reaction time (days) significant reaction with amines can be achieved 22 . In order to definitively determine the site of reaction of vinyl sulfones with 6 and 13, we employed heteronuclear multiple bond correlation (HMBC) spectroscopy, a 2D NMR technique which yields cross-peaks for 1 Figure 1 displays selected regions of HMBC spectra of phenyl vinyl sulfone derivatives, showing that 6 (panel A) forms an N7 adduct (10), while reaction with 13 (panel B) yields an N1 adduct (14) (Schemes 1 and 2, respectively). Modification of 8-thio-guanosine at N7 has been achieved by reaction with allyl bromide to form a C8-S adduct which is then transformed to the 7-allyl, 8-thioxo product via a [3,3] sigmatropic rearrangement 25 .…”