Interconversion Of Sites Research Articles

Cleavage-resistant fusion proteins of the M2 muscarinic receptor and Gαi1. Homotropic and heterotropic effects in the binding of ligands

BackgroundG protein-coupled receptors fused to a Gα-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor–G protein complex, but their usefulness depends upon the stability of the fusion. MethodsFusion proteins of the M2 muscarinic receptor and the α-subunit of Gi1 were expressed in CHO and Sf9 cells, extracted in digitonin–cholate, and examined for their binding properties and their electrophoretic mobility on western blots. ResultsReceptor fused to native αi1 underwent proteolysis near the point of fusion to release a fragment with the mobility of αi1. The cleavage was prevented by truncation of the α-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[3H]methylscopolamine was 60% of that for [3H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine. ConclusionsA stable fusion protein of the M2 receptor and truncated αi1 resembles the native receptor–G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists. General significanceRelease of the α-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein.

Ground State Structure of F1-ATPase from Bovine Heart Mitochondria at 1.9 Aå Resolution

The structure of bovine F(1)-ATPase, crystallized in the presence of AMP-PNP and ADP, but in the absence of azide, has been determined at 1.9A resolution. This structure has been compared with the previously described structure of bovine F(1)-ATPase determined at 1.95A resolution with crystals grown under the same conditions but in the presence of azide. The two structures are extremely similar, but they differ in the nucleotides that are bound to the catalytic site in the beta(DP)-subunit. In the present structure, the nucleotide binding sites in the beta(DP)- and beta(TP)-subunits are both occupied by AMP-PNP, whereas in the earlier structure, the beta(TP) site was occupied by AMP-PNP and the beta(DP) site by ADP, where its binding is enhanced by a bound azide ion. Also, the conformation of the side chain of the catalytically important residue, alphaArg-373 differs in the beta(DP)- and beta(TP)-subunits. Thus, the structure with bound azide represents the ADP inhibited state of the enzyme, and the new structure represents a ground state intermediate in the active catalytic cycle of ATP hydrolysis.

Effects of the H2S partial pressure on the performance of bimetallic noble-metal molybdenum catalysts in simultaneous hydrogenation and hydrodesulfurization reactions

Abstract The influence of the H 2 S concentration on the activity and selectivity to hydrodesulfurization (HDS) of bimetallic noble-metal molybdenum (NM–Mo, NM = Pt, Ru, Pd) supported on γ-Al 2 O 3 catalysts was studied. The effect of H 2 S on the modification of the HDS pathway and the influence of the Mo loading on the sulfur resistance of catalysts were also analyzed. NM–Mo/γ-Al 2 O 3 and conventional CoMo/γ-Al 2 O 3 catalysts were prepared and evaluated in simultaneous dibenzothiophene (DBT) HDS and naphthalene hydrogenation (HYD) reaction cycles, in the presence of dimethyl disulfide (DMDS) in order to obtain H 2 S partial pressures of 10, 30 and 50 kPa. Catalysts were characterized by atomic absorption spectroscopy and temperature-programmed reduction of sulfides (TPRS). TPRS experiments after exposing catalysts to simulated reaction conditions (TPRS-SRC) were also performed. Results show that the H 2 S does not poison the studied catalysts, but an inhibition of both HYD and HDS reactions is observed. The inhibition of catalyst activities is attributed to the interconversion of HDS, HYD and inactive sites; this interconversion is influenced by the H 2 S/H 2 ratio, the nature of the NM and the composition of catalysts. Activity, TPRS and TPRS-SRC results confirmed this scheme. A modification of the reaction pathway was observed when the H 2 S partial pressure was increased; the direct desulfurization was principally affected. The interconversion of sites could explain this modification. Moreover, our results show that the Mo loading and specifically the Pt/Mo ratio, which determine the Pt dispersion, influence the sulfur resistance of the catalyst.

Modeling in the development of hydrotreatment processes

The kinetic modeling of the hydrodesulfurization (HDS) process is based upon the detailed reaction network, distinguishes between the hydrogenolysis- and hydrogenation-steps and accounts for the adsorption of the various reacting species on two types of active sites. The functional form of the rate equations for the conversion of thiophene, (substituted) benzothiophene, (substituted) dibenzothiophene on Co/Mo-alumina appears to be identical. The nature of the active sites, the synergetic effect of Co-sulfides on the activity and the interconversion of sites by hydrogen and hydrogen sulfide are discussed. The application of rate equations accounting explicitly for the in situ conditioning of the catalyst and the HDS is illustrated for thiophene conversion. In the kinetic modeling of HDS of complex mixtures like light cycle oil, containing hundreds of S-components, the structural contribution approach is introduced to reduce the number of independent rate parameters. The structural contributions multiply the rate and adsorption parameters of the parent (unsubstituted) molecules and express the effect of the substituents. Their values were determined from experimental data. Commercial reactor operation for HDS of a complex mixture was simulated, including the effects of external and internal diffusion limitations. An example illustrates the gain that can be achieved in the removal of some of the most refractory S-components by intermediate flashing of H 2S.

Cardiac muscarinic receptors. Cooperativity as the basis for multiple states of affinity.

Cooperativity has been investigated as the mechanistic basis for effects observed with cardiac muscarinic receptors in washed membranes from Syrian hamsters. Specifically, N-[3H]methylscopolamine labeled only 66-75% of the sites labeled by [3H]quinuclidinylbenzilate at apparently saturating concentrations of each radioligand. Also, receptors labeled by N-[3H]methylscopolamine revealed three states of affinity for agonists, both in native membranes and following irreversible blockade of about 80% of the sites by propylbenzilylcholine mustard; in both preparations, guanylylimidodiphosphate (GMP-PNP) effected an apparent interconversion of sites from higher to lower affinity for agonists and from lower to higher affinity for the antagonist. Excellent and mechanistically consistent descriptions of the data were obtained in terms of a model comprising cooperative and noncooperative forms of the receptor; the former was described by a variant of the Adair equation, and the latter was included to account for low-affinity sites that survived treatment with the mustard. If differences in apparent capacity derive from negative cooperativity in the binding of N-[3H]methylscopolamine, the cooperative form of the receptor was at least trivalent in native membranes; otherwise, constraints imposed by the effects of GMP-PNP at the concentrations of radioligand used in the assays dictate that the cooperative form of the receptor was at least tetravalent. In contrast, a divalent receptor is sufficient with the data from alkylated membranes, in accord with the reduced likelihood of interactions between functional sites within an oligomeric array. A model is presented wherein the receptor interconverts spontaneously between two or more states differing in their cooperative properties. The effects of GMP-PNP can be rationalized as a shift in the equilibrium between the different states.

SELECTIVITY IN HDS, HDN, HDO AND HYDROCRACKING CONTRIBUTION OF REMOTE CONTROL AND OTHER NEW CONCEPTS

AbstractThis paper reviews some recent results in the field of hydrotreatment by sulfide catalysts. It comes in complement to a former review on the subject (1), by focusing more on new insights into the structure of the conventional CoMo (or NiMo) catalysts and the way they catalyze the various reactions involved. A first part constitutes an attempt to make an inventory of the types of catalytic sites: hydrogenation sites, sites active for the hydrogenolysis of carbon‐heteroatom bonds, acidic sites either on the sulfide phase (probably ‐SH groups) or situated on the support. A second part discusses the influence of the morphology of the MoS2crystallites, in particular the possibility that different catalytic functions be located on the rims and on the lateral edges. The variation in selectivity of the hydrotreatment catalysts as a function of the reaction conditions and their relative stability when the catalysts ages point to factors which are Independent of initial morphology. The role of spillover hydrogen will be illustrated by new examples of our laboratory concerning hydrodenitrogenation, on the one hand, and the dramatic increase of acidity of silica alumina (by a factor of more than 10) under the influence of spillover hydrogen emitted by a sulfided CoMo/Sio2catalyst, on the other hand. In the latter experiment, the hydrocracking of diphenyl methane served as a test reaction and the role of spillover was substantiated by H‐D exchange experiments. In a last part, some prospects offered by the new advances will be outlined with a special emphasis on the controlled Interconversion of sites due to remote control by spillover hydrogen. The new concepts can very likely be extended to many areas of hydrotreatment.

Effects of adenyl nucleotides and carbachol on cooperative interactions among G proteins.

Muscarinic agonists and adenyl nucleotides are noncompetitive modulators of sites labeled by [35S]GTP gamma S in washed cardiac membranes from Syrian golden hamsters. Specific binding of the radioligand and its inhibition by either GTP gamma S or GDP reveals three states of affinity for guanyl nucleotides. In the absence of adenyl nucleotide, carbachol promotes an apparent interconversion of sites from higher to lower affinity for GDP; the effect recalls that of guanyl nucleotides on the binding of agonists to muscarinic receptors. In the presence of 0.1 mM ATP gamma S, the binding of [35S]GTP gamma S is increased at concentrations up to about 50 nM and decreased at higher concentrations. At a radioligand concentration of 160 pM, binding exhibits a bell-shaped dependence on the concentration of both ATP gamma S and AMP-PNP; with ADP and ATP, there is a second increase in bound [35S]GTP gamma S at the highest concentrations of adenyl nucleotide. ATP gamma S and AMP-PNP also modulate the effect of GDP, which itself emerges as a cooperative process: that is, binding of the radioligand in the presence of AMP-PNP exhibits a bell-shaped dependence on the concentration of GDP; moreover, the GDP-dependent increase in bound [35S]GTP gamma S is enhanced by carbachol. The interactions among GDP, GTP gamma S, and carbachol can be rationalized quantitatively in terms of a cooperative model involving two sites tentatively identified as G proteins. Both GTP gamma S and GDP exhibit negative homotropic cooperativity; carbachol enhances the homotropic cooperativity of GDP and induces or enhances positive heterotropic cooperativity between GDP and [35S]GTP gamma S. An analogous mechanism may underlie the guanyl nucleotide-dependent binding of agonists to muscarinic receptors. The data suggest that the binding properties of G proteins and their associated receptors reflect cooperative effects within heterooligomeric arrays; agonist-induced changes in cooperativity may facilitate the exchange of GTP for bound GDP and thereby constitute the mechanism of G protein activation in vivo.

High-energy multiple site splitting of triplet states of aryl cations

Certain triplet-state aryl cations in microcrystalline media display site-splitting of considerable energy (0.07–0.10 cm −1). The different site species respond quite differently to warming above 77 K, and the decay kinetics between 90 and 100 K indicate some interconversion of sites. The broad triplet ESR spectra found in glassv media appear to be envelopes of the much narrower spectra obtained in microcrystals.