Cooperativity of lectin binding to lymphocytes,and its relevance to mitogenic stimulation

The relationship between the binding patterns of soybean agglutinin, peanut agglutinin (both in their native (unaggregated) form and in their polymerized form), and of Phaseolus vulgaris leucoagglutinin, to neuraminidase-treated lymphocytes from different sources, and the mitogenic activity of these lectins, was studied. In all cases investigated, binding of a lectin to lymphocytes which resulted in stimulation was a positive cooperative process. Our findings support the assumption that clustering of receptors and conformational changes in membrane structure are prerequisites for mitogenic stimulation.     

Discrimination of three parallel pathways of lactate transport in the human erythrocyte membrane by inhibitors and kinetic properties

The transmembrane movements of lactate and other monocarboxylate anions in mammalian erythrocytes have been claimed, by virtue of their sensitivity to SH-reagents, to involve a transfer system different from the classical anion system (Deuticke, B., Rickert, I. and Beyer, E. (1978) Biochim. Biophys. Acta 507, 137–155). Inhibition of monocarboxylate transfer by SH-reagents, however, was incomplete to an extent varying for different monocarboxylates. The transport component insensitive to SH-reagents has now been shown to involve (a) the classical anion-exchange system, as demonstrated by sensitivity to specific disulfonate inhibitors, and (b) nonionic diffusion, as indicated by the characteristic pH- and concentration dependency of this component and its stimulation by aliphatic alcohols. Under physiological conditions about 90% of total lactate movement proceed via the specific system, 5% via the classical anion-transfer system, 5% by nonionic diffusion. These three components of lactate exchange differ in their activation energies. The specific lactate system mediates net fluxes almost as fast as exchange fluxes, in marked contrast to the classical anion-exchange system which mediates halide exchange much faster than halide net movements. The underlying mechanism, for maintenance of electroneutrality, is an OH^?-antiport or an H⁺-symport as indicated by the particular response of lactate net fluxes to changes of intra- or extracellular pH.     

Deconvoluting the biology and druggability of protein lipidation using chemical proteomics

Lipids are indispensable cellular building blocks, and their post-translational attachment to proteins makes them important regulators of many biological processes. Dysfunction of protein lipidation is also implicated in many pathological states, yet its systematic analysis presents significant challenges. Thanks to innovations in chemical proteomics, lipidation can now be readily studied by metabolic tagging using functionalized lipid analogs, enabling global profiling of lipidated substrates using mass spectrometry. This has spearheaded the first deconvolution of their full scope in a range of contexts, from cells to pathogens and multicellular organisms. Protein N-myristoylation, S-acylation, and S-prenylation are the most well-studied lipid post-translational modifications because of their extensive contribution to the regulation of diverse cellular processes. In this review, we focus on recent advances in the study of these post-translational modifications, with an emphasis on how novel mass spectrometry methods have elucidated their roles in fundamental biological processes.     

Quantification of thiols and disulfides

Background

Disulfide bond formation is a key posttranslational modification, with implications for structure, function and stability of numerous proteins. While disulfide bond formation is a necessary and essential process for many proteins, it is deleterious and disruptive for others. Cells go to great lengths to regulate thiol-disulfide bond homeostasis, typically with several, apparently redundant, systems working in parallel. Dissecting the extent of oxidation and reduction of disulfides is an ongoing challenge due, in part, to the facility of thiol/disulfide exchange reactions.

Scope of review

In the present account, we briefly survey the toolbox available to the experimentalist for the chemical determination of thiols and disulfides. We have chosen to focus on the key chemical aspects of current methodology, together with identifying potential difficulties inherent in their experimental implementation.

Major conclusions

While many reagents have been described for the measurement and manipulation of the redox status of thiols and disulfides, a number of these methods remain underutilized. The ability to effectively quantify changes in redox conditions in living cells presents a continuing challenge.

General significance

Many unresolved questions in the metabolic interconversion of thiols and disulfides remain. For example, while pool sizes of redox pairs and their intracellular distribution are being uncovered, very little is known about the flux in thiol-disulfide exchange pathways. New tools are needed to address this important aspect of cellular metabolism. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin

Gurmarin is a 35 amino acid peptide with three disulfide bridges in an inhibitor cystine knot. It is found in the plant Gymnema sylvestre, and has been identified as a sweet taste inhibitor in rodents. In this article we provide an efficient route for the synthesis of gurmarin by a controlled random oxidation strategy. We compared two oxidation procedures to form the three disulfide bridges. In the first, based on random oxidation, reduced gurmarin was synthesized using trityl for cysteine protection, and oxidized for 48h in a Tris-HCl buffer containing cystamine and reduced glutathione to facilitate disulfide scrambling. The second was based on step-wise deprotection followed by oxidation in which the cysteine pairs are orthogonally protected with tert-Butylthio, trityl and acetamidomethyl. To verify that the native gurmarin oxidation product was obtained, thermolysin cleavage was used. Cleavage of random oxidized gurmarin showed two possible disulfide combinations; the native and a non-native gurmarin disulfide isomer. The non-native isomer was therefore synthesized using the orthogonal deprotection-oxidation strategy and the native and the non-native gurmarin isomers were analyzed using UPLC. It was found that the random oxidation procedure leads to native gurmarin in high yield. Thus, the synthetic route was simple and significantly more efficient than previously reported syntheses of gurmarin and other cysteine rich peptides. Importantly, native gurmarin was obtained by random oxidation, which was confirmed by a synthetic approach for the first time.     

Formation of aqueous pores in the human erythrocyte membrane after oxidative cross-linking of spectrin by diamide

Oxidation of erythrocyte membrane SH-groups by diamide and tetrathionate induces cross-linking of spectrin (Haest, C.W.M., Kamp, D., Plasa, G. and Deuticke, B. (1977) Biochim. Biophys. Acta 469, 226–230). This cross-linking was now shown to go along with a concentration- and time-dependent enhancement of membrane permeability for hydrophilic nonelectrolytes and ions. The enhancement is specific for oxidative SH-group modifications, is reversible by reduction of the induced disulfides, can be suppressed by a very brief pre-treatment of the cells with low concentrations of $\text{N-}\text{ethylmaleimide}$ and is strongly temperature-dependent. The pathway of the induced permeability discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy ($\text{E}{}_{\mathrm{<mtext>a</mtext>}}\text{3–8}\text{kcal/mol}$). These findings are reconcilable with the formation of a somewhat inhomogeneous population of aqueous pores with radii probably $\text{? 0.65}\text{nm}$. Estimated pore numbers vary with the size of the probe molecule. Assuming a diffusion coefficient as in bulk water within the pore, at least 20 pores per cell have to be postulated; more realistic lower diffusion coefficients increase that number. Alterations of the lipid domain by changes of cholesterol contents and insertion of hexanol or nonionic detergents alter the number or size of the pores. Since aggregation of skeletal and intrinsic membrane proteins also occurs after the SH-oxidation, in parallel to the formation of membrane leaks, one may consider (a) defects in the disturbed bilayer interface, (b) a mismatch between lipid and intrinsic proteins or (c) channels inbetween aggregated intrinsic proteins as structures forming the pores induced by diamide treatment.     

Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry

Alkylation converts Cys thiols to thioethers and prevents unwanted side reactions, thus facilitating mass spectrometric identification of Cys-containing peptides. Alkylation occurs preferentially at Cys due to its high nucleophilicity, however reactions at other such sites are possible. N-ethylmaleimide (NEM) shows rapid reaction kinetics with Cys and careful definition of reaction conditions results in little reactivity at other sites. Analysis of a protein standard alkylated under differing reaction conditions (pH, NEM concentrations and reaction times) was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and selected reaction monitoring (SRM) of NEM-modified and unmodified peptide pairs. Mis-alkylation sites at primary and secondary amines were identified and limited to one equivalent of NEM. No evidence for hydroxyl or thioether alkylation was observed. Improved specificity was achieved by restricting the pH below neutral, NEM concentration below 10 mM and/or reaction time to below 5 min. Maximal removal of Cys activity was observed in tissue homogenates at 40 mM NEM within 1 min, dependent upon efficient protein denaturation. SRM assays identified peptide-specific levels of mis-alkylation, indicating that NEM-modified to unmodified ratios did not exceed 10%, with the exception of Cys alkylation that proceeded to 100%, and some Lys residues that resulted in tryptic missed cleavages. High reactivity was observed for His residues considering their relatively low abundance. These data indicate that rapid and specific Cys alkylation is possible with NEM under relatively mild conditions, with more abrasive conditions leading to increased non-specific alkylation without appreciable benefit for MS-based proteomics.     

Sulfhydryl group modification of photoreceptor G-protein prevents its light-induced binding to rhodopsin

The effect of sulfhydryl modification on the light-induced interaction between rhodopsin and the peripheral GTP-binding protein of the photoreceptor membrane (G-protein) has been investigated by time-resolved near-infrared light-scattering and polyacrylamide gel electrophoresis. It has been found that the modification of rhodopsin with the alkylating agent N-ethylmaleimide (NEM) does not affect its light-induced interaction with the G-protein. Modification of G-protein with NEM or other sulfhydryl agents prevents any light-induced binding to rhodopsin. Dark-association of G to the membrane as well as the light-induced complex with rhodopsin (once formed) is insensitive to NEM.     

Characterization of ATP-Sensitive Potassium Channels Functionally Expressed in Pituitary GH3 Cells

ATP-sensitive K⁺ (K_ATP) channels have been characterized in pituitary GH₃ cells with the aid of the patch-clamp technique. In the cell-attached configuration, the presence of diazoxide (100 μm) revealed the presence of glibenclamide-sensitive K_ATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mm) or sodium cyanide (300 μm) increased K_ATP channel activity, while nicorandil (100 μm) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of K_ATP channels in a concentration-dependent manner with an IC₅₀ value of 30 μm. The activation of phospholipase A₂ caused by mellitin (1 μm) was found to enhance K_ATP channel activity and further application of aristolochic acid (30 μm) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4′-dithiodipyridine (100 μm) also induced K_ATP channel activity. Diazoxide, mellitin and 4,4′-dithiodipyridine activated the K_ATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of diazoxide (100 μm) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly indicates that K_ATP channels similar to those seen in pancreatic β cells are functionally expressed in GH₃ cells. In addition to the presence of Ca²⁺-activated K⁺ channels, K_ATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane potential. Received: 19 June 2000/Revised: 13 September 2000     

The effect of metal ions on mitochondrial pyridine dinucleotide transhydrogenase

Bovine heart submitochondrial particle transhydrogenase is inhibited by cations in a concentration and pH-dependent manner, and non-energy-linked transhydrogenation is inhibited to a greater extent by metals than the energy-linked reaction. The inhibition of the enzyme by Mg²⁺ is competitive with the NADP substrate and non-competitive with the NAD substrate. Mg²⁺ stimulates inactivation of the enzyme by 5,5′-dithiobis(2-nitrobenzoic acid), and protects against thermal and proteolytic inactivation. This suggests that Mg²⁺ binding in the NADP site alters transhydrogenase to a more thermostable conformation, which is less susceptible to attack by trypsin and more reactive with 5,5′-dithiobis(2-nitrobenzoic acid). Other cation inhibitors mimic Mg²⁺ in these properties. The order of effectiveness of the inhibitors tested is La³⁺ > Mn²⁺ > Ca²⁺ ? Mg²⁺ > Sr²⁺ > Na⁺ ? K⁺. This order is described by the Irving-Williams order for the stability of metal-ligand complexes, suggesting that carboxylates or amines may comprise the inhibitory cation binding site.