Bernard Foucaud

Cysteine mutants as chemical sensors for ligand-receptor interactions.

The incorporation of cysteine residues into membrane receptors by mutagenesis has enabled the development of engineered proteins. Chemical modification of the mutant receptor using a wide range of biochemical and biophysical probes has facilitated functional studies of ligand-receptor interactions. In particular, the substituted-cysteine accessibility method (SCAM) represents a successful example of how to probe transmembrane receptor domains after chemical modification of the mutants with sulfydryl-reacting molecules. We propose an extension of this methodology using site-specific affinity probes that react with cysteine mutants to gain reliable structural information on the binding of a ligand in its receptor site.

Kinetic and morphological analysis of the preferential adhesion of chick embryo neuronal cells to astroglial cells in culture

Abstract Dissociated cells (mainly neuroblasts) obtained from brain hemispheres of chick embryos adhere to a preformed layer of chick brain astroglial cells faster and to a greater extent than to a layer of chick fibroblasts. At the begining of the experiment the adhered neuroblasts are dispersed on both layers but within a short time neuroblasts on a fibroblast layer migrate, form clusters and only later on begin to differentiate; in contrast those on an astroglial layer remain dispersed and differentiate rapidly. In both cases a number of cells of each inoculum do not adhere to either cellular layer, but these cells do not differ morphologically from those which adhered. Adhesion of neuroblasts to astroglial layers and that to fibroblast layers appear to follow the kinetics of an irreversible one-step reaction in pseudo-first-order conditions only for the first 50 and 60 min respectively. The departure from linearity of the kinetics after these times does not correspond, however, to the occurrence of second-order conditions. Other possible mechanisms of adhesion which could explain the experimental data are compared with those expected for different kinetic mechanisms. A model in which cell adhesion is treated in terms of affinity between receptor and ligands is used to analyse the nature of cell-cell adhesion.

Preparation of 3-chloroacetylpyridine adenine dinucleotide: An alkylating analogue of NAD+

Analogues of coenzymes with reactive groups have been extensively used to detect the presence of functional amino acid residues in the catalytic site of dehydrogenases. Thus, alkylating analogues of NAD+ have been synthesized: the group at C-3 of nicotinamide of NAD+ has been replaced by a diazonium group [ 11, a diazoacetate [2] or a bromoacetyl group, with an aliphatic chain instead of the ribose [3-61 and the adenine has been replaced by a bromoacetylimidazole [6, 71. As basic residues are invoked in the mechanism of the dehydrogenase for the removal of the proton located at the alcohol during the hydrogen transfer [8,9], it was tempting to attack this basic group from the coenzyme side, by the chloroacetyl group, replacing at C-3 the amide group of the pyridinium ring. Our goal was to synthesize the 3-chloroacetylpyridine adenine dinucleotide analogue of NAD+. This is quite similar to 3-acetylpyridine adenine dinucleotide, which is active as hydrogen acceptor with many dehydrogenases. The similarity of the structure may lead to the activity of this analogue as hydrogen acceptor, as well as to alkylation. This activity is therefore a good indication for the alkylation of a residue in the active site. The preparation of this analogue, and of the 3-propionylpyridine adenine dinucleotide are described here and the preliminary results on the alkylation of some dehydrogenases are briefly reported.

Comparative binding properties of linear and cyclic δ-selective enkephalin analogues: [3H]-[D-Thr2, Leu5] enkephalyl-Thr6 and [3H]-[D-Pen2, D-Pen5] enkephalin

The range of δ‐selectivity of linear and cyclic analogues of enkephalin in rat brain was found to be: [D‐P n5] enkephalin (DPLPE) > [D‐P n5] enkephalin (DPDPE) > [D‐Thr2, Leu5] enkephalyl‐Thr6 (DTLET) > [D‐Ser2, Leu5] enkephalyl‐Thr6 (DSLET). Saturation experiments performed with [3H]DPDPE and [3H]DTLET in NG108‐15 cells and rat brain showed similar binding capacities for both the ligands, but the δ‐affinity of [3H]DTLET (K D ≈ 1.2 nM) was much better than that of [3H]DPDPE (K D ≈ 7.2 nM). The rather low δ‐affinity of DPDPE induced high experimental errors cancelling the benefit of its better δ‐selectivity. Binding experiments in rat or guinea‐pig brains showed, in both cases, the better δ‐selectivity of [3h]dtlet compared to [3h]slet. The former peptide remains at this time the most appropriate radioactive probe for binding studies of δ‐receptor.

Reactive derivatives for affinity labeling in the ifenprodil site of NMDA receptors.

To prepare thiol-reactive ifenprodil derivatives designed as potential probes for cysteine-substituted NR2B containing NMDA receptors, electrophilic centers were introduced in different areas of the ifenprodil structure. Intermediates and final compounds were evaluated by binding studies and by electrophysiology to determine the structural requirements for their selectivity. The reactive compounds were further tested for their stability and for their reactivity in model reactions; some were found suitable as structural probes to investigate the binding site and the docking mode of ifenprodil in the NR2B subunit.

Role of thiol group(s) in the enzymatic activity of the membrane-bound Δ5-3β-hydroxysteroid dehydrogenase from bovine adrenal cortex microsomes

Abstract The effect of different sizes of thiol groups reagents on the kinetic parameters of the membrane-bound Δ 5 -3β- hydroxy-steroid dehydrogenase (3(or 17)-β-hydroxysteroid:NAD(P)+ oxidoreductase, EC 1.1.1.51) from bovine adrenal cortex microsomes was examined. Iodoacetamide and parachloromercuribenzoate increased the maximum velocity 1.6 times and also the K m for adrost-5-en-3β-ol-17-one by factors of 1.5 and 5.5, respectively. Hydrophobic reagents of larger size led to a large decrease of the maximum velocity and further increase in the K m for adrost-5-en-3β-ol-17-one. Androst-5-en-3β-ol-17-one hindered the activating effect of iodoacetamide. Similar results were found with the NAD+ analogue, 3-chloro-acetyl pyridinium adenine dinucleotide, an electrophilic reagent. A decrease of the maximum velocity was observed (38% inhibition at the highest concentration used). As with other SH reagents, the K m for androst-5-en-3β-ol-17-one was increased 5 times. Preincubation of membranes with iodoacetamide prevented the enzyme from inhibition by 3-chloroacetyl pyridinium adenine dinucleotide. Protection experiments with androst-5-en-3β-ol-17-one, oestr-1,3,5(10)6,8-pentaen-3-ol-17-one and NAD+ demonstrated that the steroids were more effective than NAD+ in preventing the enzyme from inhibition. In all cases, the K m for NAD+ was unaltered. From these results, it is suggested that thiol reagents, including 3-chloroacetyl pyridium adenine dinucleotide, substitute an SH group in the vicinity of the steroid-binding site of the enzyme.

Properties of horse liver alcohol dehydrogenase modified by the affinity label 3-chloroacetylpyridine-adenine dinucleotide.

The reactive analogue of NAD+, CPAD+, was incorporated in the horse liver alcohol dehydrogenase (EC 1.1.1.1) linearly with its inactivation, to stoichiometry, with no apparent subunit interaction. No hydride transfer could take place in the modified enzyme, nor the interaction of trans-4-N,N-dimethylaminocinnamaldehyde with its reduced form, indicating an impairment of the accessibility to the catalytic zinc atom. The labeling in the enzyme, alkylated by [carbonyl-14C]CPAD+ was not stable, with a half-life of 32 h.

Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide.

Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett. 40, 29-32). Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label. The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site. The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants. In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified. Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.

A photoaffinity probe for the polyamine site regulating the NMDA receptor.

Polyamines produce on the NMDA-receptor channel activity a regulatory effect subsequent to their binding to specific sites on the receptor-complex. The photoactivatable polyamine derivative L-azidophenylspermine shows properties which suggest its potential as a photoaffinity probe to investigate the nature and topology of these sites. In the dark, its effect on the binding of tritiated N-(1-[thienyl]cyclohexyl)piperidine ([3H]TCP) to synaptosomal plasma membranes is similar to that of diaminodecane. Arcaine antagonizes both the effects of L-azidophenylspermine and diaminodecane on [3H]TCP binding. L-Azidophenylspermine competes in post-synaptic densities with tritiated spermidine for a unique binding site with an EC50 similar to that of spermine. Upon irradiation, L-azidophenyl-spermine incorporates into this material with a high efficiency to a level consistent with both the Bmax for tritiated spermidine and the estimated density of NMDA receptors in this fraction.

Equilibrium-driven mechanism for preferential adhesion between chick embryo cells

The experiments presented here confirm the hypothesis according to which, in our experimental system of differential cell adhesion (where we studied the kinetics of the earliest period of adhesion of a suspension of chick embryo neuroblasts to layers of astroblasts or fibroblasts), the mechanism of adhesion appears to consist of two steps, the first of which is a short-term reversible phase corresponding to a binding equilibrium. In fact, adhesion of neuroblasts to each of the two cell layers occurs according to kinetic constants and attains levels which are characteristic for each of the two adhesion systems. In both systems, neuroblasts that have not adhered at equilibrium are able to adhere if inoculated over a fresh cell layer of the same type, as they do during the first inoculation; conversely, neuroblasts that have adhered to a cell layer can be made to de-adhere by substituting cell-free fresh medium to the inoculation medium containing non-adhering neuroblasts. This shows that, as predicted for a reversible equilibrium system, removal of adhering neuroblasts from the system at equilibrium provokes adhesion, and removal of non-adhered neuroblasts provokes de-adhesion. Furthermore the level of adhesion at equilibrium is, in all cases, the same. The reversibility of adhesion, which is almost quantitative during the onset of the equilibrium, gradually decreases with time, indicating the presence of a process of irreversible attachment between cells after the first reversible step. The developmental implications of the complete sequential mechanisms are briefly discussed.