On the, stoichiometry between uncouplers of oxidative phosphorylation and respiratory chains. The catalytic action of SF 6847 (3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile)

Titration of State 4 rat-liver mitochondria at pH 7.2 with the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) at various concentrations of mitochondria and using various substrates indicates that under optimal conditions less than 0.2 molecule of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile per respiratory chain is sufficient to induce complete uncoupling. This result suggests that there is not a stoichiometric relationship between uncoupler molecules and cytochrome c oxidase, involved in oxidative phosphorylation, or between the former and phosphorylation assemblies.

Experiments on the release by 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile of azide-inhibited respiration of State 3 mitochondria and titrations with 5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanilide (S₁₃) of State 4 mitochondria at various mitochondrial concentrations confirm this conclusion.     

Inhibition of cobalt(II) carboxypeptidase A by ligands. Kinetic evidence for the formation of a ternary enzyme-metal-ligand complex

Saturation kinetics are observed in the inhibition of cobalt carboxypeptidase A by the chelating agent 1,10-phenanthroline. The association constant K₁ for the formation of the enzyme-metal-ligand ternary complex and k₂, the rate of breakup of the ternary complex, have been obtained. A mechanism is proposed to account for the pH profile of the reaction which, in conjunction with K₁, permits the calculation of the individual rate constants k₁, K_?1, k₂, k₃. The magnitude of the rate constant k₁ suggests that cobalt(II) in CoCPA is five-coordinate. Similar but less extensive studies on inhibition by 2,2′-bipyridyl and 8-hydroquinoline-5-sulfonic acid have also been carried out     

Direct evidence of a heterotrimeric complex of human interleukin-4 with its receptors.

The mode of binding of interleukin-4 (IL-4) to its two known receptors, specific receptor IL-4R and a shared receptor gamma c, was investigated using gel filtration and gel electrophoresis. A ternary complex between IL-4 and the soluble domains of the two receptors was shown to exist in solution. The association constant between gamma c and the stable complex of IL-4/sIL-4R is in the millimolar range, making the ternary complex a feasible target for crystallization studies.     

Structure of aldehyde reductase in ternary complex with coenzyme and the potent 20alpha-hydroxysteroid dehydrogenase inhibitor 3,5-dichlorosalicylic acid: implications for inhibitor binding and selectivity

The structure of aldehyde reductase (ALR1) in ternary complex with the coenzyme NADPH and 3,5-dichlorosalicylic acid (DCL), a potent inhibitor of human 20α-hydroxysteroid dehydrogenase (AKR1C1), was determined at a resolution of 2.41 Å. The inhibitor formed a network of hydrogen bonds with the active site residues Trp22, Tyr50, His113, Trp114 and Arg312. Molecular modelling calculations together with inhibitory activity measurements indicated that DCL was a less potent inhibitor of ALR1 (256-fold) when compared to AKR1C1. In AKR1C1, the inhibitor formed a 10-fold stronger binding interaction with the catalytic residue (Tyr55), non-conserved hydrogen bonding interaction with His222, and additional van der Waals contacts with the non-conserved C-terminal residues Leu306, Leu308 and Phe311 that contribute to the inhibitor’s selectivity advantage for AKR1C1 over ALR1.     

Interrogation of a Sonogashira cross-coupling of 8-bromoguanosine with phenylacetylene on amberlite: evidence for Pd/Cu ion binding and propagation of Pd/Cu nanoparticles

The reactivity of Amberlite (IRA-67) base "heterogeneous" resin in Sonogashira cross-coupling of 8-bromoguanosine 1 with phenylacetylene 3 to give 2 has been examined. Both 1 and 2 coordinate to Pd and Cu ions, which explains why at equivalent catalyst loadings, the homogeneous reaction employing triethylamine base is poor yielding. X-ray photo-electron spectroscopy (XPS) has been used to probe and quantify the active nitrogen base sites of the Amberlite resin, and postreaction Pd and Cu species. The PdCl(2)(PPh(3))(2) precatalyst and CuI cocatalyst degrade to give Amberlite-supported metal nanoparticles (average size ～2.7 nm). The guanosine product 2 formed using the Amberlite Pd/Cu catalyst system is of higher purity than reactions using a homogeneous Pd precatalyst, a prerequisite for use in biological applications.     

Reactions of Cr(III) tetraphenylporphyrin complex with superoxide ion: ESR evidence for the formation of a new superoxide adduct of Cr(IV) porphyrinate and its reactive character

The electron spin resonance (ESR) spectrum of the reaction product of superoxide ion, O₂⁻, with chloro-5,10,15,20-tetraphenylporphyrinatochromium(III) [Cr(III)(TPP)Cl] shows strong hyperfine interactions with the metal nucleus and the metal ligand, indicating the formation of a superoxide adduct, Cr(IV)(TPP)(Cl)(O₂⁻). The formation of this superoxide adduct was also confirmed by UV-Vis spectroscopy. The reactive character of this superoxide adduct was investigated by ESR spectrometry. It was found that Cr(IV)(TPP)(Cl)(O₂⁻) can oxidize t-butylamine and triphenylphosphine to give the corresponding radical species, respectively, but not pyridine, 4-cyanopyridine or imidazole. These results indicate that the reactive character of Cr(IV)(TPP)- (Cl)(O₂⁻) resembles that of the free superoxide ion.     

RICH-1 has a BIN/Amphiphysin/Rvsp domain responsible for binding to membrane lipids and tubulation of liposomes

RhoGAP interacting with CIP4 homologs-1 (RICH-1) was previously found in a yeast two-hybrid screen for proteins interacting with the SH3 domain of the Cdc42-interacting protein 4 (CIP4). RICH-1 was shown to be a RhoGAP for Cdc42 and Rac. In this study, we show that the BIN/Amphiphysin/Rvsp (BAR) domain in RICH-1 confers binding to membrane lipids, and has the potential to deform spherical liposomes into tubes. In accordance with previous findings for the BAR domains in endophilin and amphiphysin, RICH-1-induced tubes appeared striated. We propose that these striated structures are formed by oligomerization of RICH-1 through a putative coiled-coil region within the BAR domain. In support of this notion, we show that RICH-1 forms oligomers in the presence of the chemical cross-linker BS3. These results point to an involvement of RICH-1 in membrane deformation events.     

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: evidence for a novel Ap4A synthetic activity

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATP. Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap4A bound to the active site pocket suggesting the presence of Ap4A synthetic activity in TdcD. Binding of Ap4A to the enzyme was confirmed by the structure determination of a TdcD-Ap4A complex obtained after cocrystallization of TdcD with commercially available Ap4A. Mass spectroscopic studies provided further evidence for the formation of Ap4A by propionate kinase in the presence of ATP. In the TdcD-Ap4A complex structure, Ap4A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction.     

The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 A resolution

The complex formed by bovine trypsinogen and the pancreatic trypsin inhibitor crystallizes in large crystals isomorphous with trypsin-PTI² complex crystals Rühlmann et al. 1973. X-ray diffraction data to 1.9 Å resolution were collected in the absence and presence of Ile-Val dipeptide. Both trypsinogen complex structures have been crystallographically refined, using the refined trypsin-PTI complex Huber et al. 1974a as a starting model. The final R values are 0.25 and 0.26, respectively. The mean main-chain atom deviations between the three complex structures are about 0.15 Å. In contrast, the mean deviation between the complexed and the free trypsinogen Fehlhammer et al. 1977 is 0.28 Å, reflecting the influence of crystal packing and complexation. The trypsinogen component adopts a trypsin-like conformation upon PTI binding: The Asp194 side-chain turns around and the activation domain becomes rigid, forming the specificity pocket and the Ile16 binding cleft. The specific interactions between PTI and trypsin are also observed in the trypsinogen complex. As in free trypsinogen, the N-terminus including residues Val10 to Gly18 is mobile and sticks out into solution. Apart from the different arrangement of the N-termini in the two complexes, the only significant, but minor structural difference is the enhanced thermal mobility of the autolysis loop in the trypsinogen complex. Upon binding of the Ile-Val dipeptide, the autolysis loop becomes fixed as in the trypsin complex. The Ile-Val position is identical in the ternary and the trypsin complex.     

Minimum length of an idiotypic peptide and a model for its binding to a major histocompatibility complex class II molecule.

We have defined the minimum length of a synthetic peptide which can activate I-Ed-restricted BALB/c T cell clones specific for a mutated self-antigen: an idiotope on the syngeneic lambda 2315 immunoglobulin light chain. A peptide comprising residues 91-101 of the lambda 2315 sequence had full stimulatory potency. Surprisingly, a peptide analogue in which His97 was deleted was almost fully active. Truncated, deleted or substituted peptide analogues did not distinguish between seven T cell clones that use different alpha/beta T cell receptors. The 91-101 region in the lambda 2315 light chain does not form an amphipathic helix even though such a helix has been suggested to be important for T cell epitopes. Further, a motif proposed by Rothbard and Taylor as being common to T cell immunogenic peptides is not necessary for the lambda 2315 idiotypic peptide. Comparison with seven other I-Ed-restricted peptides revealed that the peptides are generally positively charged and have two basic amino acids clustered around the centre. On the basis of a model of the class II molecule peptide binding site, we suggest that these positively charged residues may interact with the negatively charged residues at positions 114(Glu) and 155(Asp) of the E beta d chain.     

Use of the twin-cell differential titration calorimeter for binding studies. I. EDTA and its calcium complex

The use of a twin-cell differential titration calorimeter is described which utilizes small volumes (1–3 ml) of modest concentrations of materials (0.001–0.01 M) and yield data of good precision. Operation is controlled by a microprocessor which regulates and controls the addition of reagents and collects and displays the data as time, temperature in volts, and the pH. Corrections for the titration of water are applied to the potentiometric data, and the thermal data are corrected for the initial temperature-time baseline, the changes in heat capacity, and the heat loss (or gain) to the external environment. Finally, the thermal signal is corrected for the heat derived from the formation of water due to the free hydrogen or hydroxyl ions present. The corrected data as pH, groups titrated adn ΔH_T (kcal/mol) can then be used to obtain the parameters pK′ and ΔH_i involved with the equilibria by curve-fitting the observed data.

The system has been applied to the ionization of EDTA and its calcium complex. The ionization constants, the heats of ionization, the stoichiometry of binding and the heat of binding have been determined and demonstrated to be in agreement with published values.     

Phosphatidylinositol exchange protein. Effects of membrane structure on activity and evidence for a ping-pong mechanism.

Phosphatidylinositol exchange protein, purified from bovine cerebral cortex, catalyzes the transfer of phosphatidylinositol and, to a lesser extent, phosphatidylcholine between rat liver microsomes and egg phosphatidylcholine liposomes. Transfer activity is sensitive to pH, temperature, and the method of liposome preparation. Variation of the phospholipid composition of the liposomes produces vesicles for which the apparent Michaelis constant decreases with increasing molar proportions of phosphatidylinositol. Interaction of exchange protein with liposomes containing radioactively labeled phosphatidylcholine allows the isolation of a phospholipid-protein complex; dissociation of this complex occurs upon subsequent interaction with unlabeled liposomes. Changes in the concentration of the two membrane species, microsomes and liposomes, yield results which are interpreted in terms of a ping-pong kinetic mechanism for the protein-catalyzed, intermembrane transfer of phospholipids.     

Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex

The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT > TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s(-1) to 96 s(-1). Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in the literature.     

Secondary structure of a truncated form of lecithin retinol acyltransferase in solution and evidence for its binding and hydrolytic action in monolayers

Lecithin retinol acyltransferase (LRAT) is a 230 amino acids membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. The enzymatic activity of a truncated form of LRAT (tLRAT) which contains the residues required for catalysis but which is lacking N- and C-terminal hydrophobic segments has been shown to depend on the detergent used for its solubilization. Moreover, it is unknown whether tLRAT can bind membranes in the absence of these hydrophobic segments. The present study has allowed to measure the membrane binding and hydrolytic action of tLRAT in lipid monolayers by use of polarization modulation infrared reflection absorption spectroscopy and Brewster angle microscopy. Moreover, the proportion of the secondary structure components of tLRAT was determined in three different detergents by infrared absorption spectroscopy, vibrational circular dichroism and electronic circular dichroism which allowed to explain its detergent dependent activity. In addition, the secondary structure of tLRAT in the absence of detergent was very similar to that in Triton X-100 thus suggesting that, compared to the other detergents assayed, the secondary structure of this protein is very little perturbed by this detergent.     

Giant unilamellar vesicles (GUVs) as a new tool for analysis of caspase-8/Bid-FL complex binding to cardiolipin and its functional activity

Cardiolipin (CL) has recently been shown to be both an anchor and an essential activating platform for caspase-8 on mitochondria. These platforms may be at the mitochondrial contact sites in which truncated Bid (tBid) has been demonstrated to be located. A possible role for CL is to anchor caspase-8 at contact sites (between inner and outer membranes), facilitating its self-activation, Bid-full length (FL) cleavage, tBid generation (and Bax/Bak activation and oligomerization), mitochondrial destabilization and apoptosis. We have developed an in vitro system that mimics the mitochondrial membrane contact site platform. This system involves reconstituting caspase-8, Bid-FL and CL complexes in giant unilamellar vesicles (GUVs). We first validated the system by flow cytometry analysis of light-scattering properties and nonyl acridine orange staining of their CL content. Then, we used flow cytometry analysis to detect the binding of active caspase-8 to CL and the subsequent truncation of bound Bid-FL. The tBid generated interacts with CL and induces GUV breakage and partial re-vesiculation at a smaller size. Our findings suggest an active role for mitochondrial membrane lipids, particularly CL, in binding active caspase-8 and providing a docking site for Bid-FL. This phenomenon was previously only poorly documented and substantially underestimated.     

Covalent Cross-Linking of Radiolabeled N-Formylated Hexapeptide to its Specific Receptor on Rat and Human Neutrophils: Evidence for a ligand induced complex formation

Abstract

The structure of the rat and human neutrophil receptor for N-formylated chemotactic peptides was characterized using ¹²⁵I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys hexapeptide as a ligand and an affinity cross-linking technique. ¹²⁵I-hexapeptide bound to purified rat peritoneal neutrophils was time, temperature and pH-dependent. The average receptor number per cell was about 67.000 and díssociation constant (K_d) 0.41 nM. Formyl-MLLP, fMLP, fNLP, were 750%, 15%, 8.6% respectively and Boc-MLP, Boc-NLP, and MLP 0.6% as potent as the hexapeptide in inhibiting the binding of ¹²⁵I-hexapeptide to rat neutrophils. The same correlation was found between these peptides in their potency to induce chemotaxis. This indicates that the rat neutrophil chemotactic receptor is like human receptor also a highly stereoselective and requires a N-formylated ligand for high affinity binding. Affinity cross-linking of ¹²⁵I-hexapeptide to rat and human neutrophil chemotactic receptor with glutaraldehyde revealed on SDS-PAGE a 85-kDa and 62-kDa major complex and a 170-kDa and 120-kDa minor complex, respectively. The 120-kDa complex was absent in human neutrophils if the cells were treated with glutaraldehyde prior to cross-linking of ¹²⁵I-hexapeptide to its receptor. Likewise, the larger complex was absent if neutrophils were exposed to heterelogous ligand (C5a) prior to glutaraldehyde treatment and cross-linking of ¹²⁵I-hexapeptide to its specific receptor. These results demonstrate that the rat neutrophils possess a functional high-affinity receptor for N-formylated chemotactic peptides and that the size of the monomeric receptor is 85-kDa and about 23-kDa larger than that of the human receptor. Upon homologous ligand binding the receptor seems to form a larger complex.