Max Hill

Disuccinimidyl esters as bifunctional crosslinking reagents for proteins: Assays with myosin

Bifunctional crosslinking reagents have been used in studies of the spatial arrangement of muscle contractile proteins, such as myosin and actin, either in their soluble forms [ 1,2], or in synthetic filaments [3-51, or even in myofibrils [3]. The most commonly used reagents are the bisimidates, which are very reactive, but also quite unstable in aqueous solution; incomplete substitution and unexpected side reactions furthermore occur if the crosslinking reaction is at pH -8 [6,7]. A disuccinimidyl ester, the dithiobis (succinimidyl propionate), DSP, that does not have these drawbacks and contains, moreover, an easily cleavable disulfide bond, has been described [8]; unlike the bis-imidates, this reagent allowed the crosslinking of the two heads of a myosin molecule [ 21. Owing to the high chemical reactivity of DSP and its stability in water, we thought it interesting to synthesize a series of disuccinimidyl esters of various chain lengths (table 1). These include non-cleavable reagents (compounds I-IV), and also reagents with either a uic-glycol (compounds V, VI) or an ethylenic bond (compound VII); the crosslinks formed by these last compounds can in principle be cleaved,

Octameric structure of yeast phosphofructokinase as determined by crosslinking with disuccinimidyl β‐hydromuconate

Yeast phosphofructo~nase (EC 2.7.1.1 l), a key enzyme in the regulation of the glycolytic pathway, is built up of an equal number of two types of subunits (Y and /3 which are immunologically distinct [l] and further differentiated by their relative susceptibility to proteolytic degradation in the presence of specific ligands [2]. Binding experiments [3,4] as well as functional properties of yeast phosphofructokinase [4,5] were indicative of a small number of interacting protomers (3 or 4) equal to only half the number of subunits constituting the enzyme oligomer. However, quaternary structure of yeast phosphofructokinase is still a matter of controversy since molecular weight determinations were unable to discriminate unambiguously between a hexameric and an octameric structure of the enzyme [4,6,7]. Thus, the major evidence for an octameric structure was obtained from small-angle X-ray scattering [S] and from crosshnking experiments with dimethyl suberimidate [9]. However, these data could not rule out an eventual hexameric structure for yeast phosphofructokinase. Indeed, the interpretation of X-ray diffusion data relies heavily on the assumption of an arbitrary chosen shape of the subunits. Moreover, no polymeric forms higher than tetramer

ATP:AMP phosphotransferase activity, a new characteristic of Catharanthus roseus tonoplasts

The ATPase activity of Catharanthus roseus tonoplasts was examined using HPLC separation and quantification of adenine nucleotides. ATP seemed to be degraded into ADP and AMP by tonoplast vesicles. When ADP was the initial substrate, the appearance of AMP and concomitant ATP synthesis were observed; these reactions were inhibited by Ap5A. The apparent degradation of ATP into AMP was also inhibited by Ap5A. These results indicated that AMP arose from an ATP:AMP phosphotransferase activity and excluded the possibility of the hydrolysis of ADP into AMP by the tonoplast ATPase. AMP was degraded by the microsomal fraction from protoplasts or by the cytosol while the tonoplast vesicles did not hydrolyze it. This observation was used to assess the purity of tonoplasts.

Luminescence of the tryptophan and tyrosine residues of trypsin

Summary The fluorescence and the phosphorescence of bovine trypsin have been investigated. The emission spectra of the three forms of the enzyme do not reveal any influence of the intrachain splits on the environment of the tryptophan residues. The particular importance of the first split (Lys Ser132) is shown by the investigation of the denaturation of β, α and ψ131 trypsin by dissociating reagents like 8 M urea and 0.5 per cent sodium dodecylsulfate. The binding of competitive inhibitors and the thermal fluorescence transitions show that some tryptophan residues are involved in the conformational changes of the substrate binding region of trypsin.

Does a proton-pumping ATPase exist in the tonoplast?

In order to account for the accumulation of metabolites in plant vacuoles, the existence of a proton-pumping ATPase has been widely suggested in the literature. The demonstration of such a tonoplast-bound ATPase was merely based on the characterization of a nitrate-sensitive microsomal fraction. In some examples, this ATPase activity has been evidenced on vacuole preparations obtained under conditions which were criticized by Boller. The application of the reverse phase high-performance liquid chromatography method (RP-HPLC) to the simultaneous separation of adenine nucleotides, in the presence of tonoplast vesicles isolated from Catharanthus roseus, showed results not necessarily correlated with the ATPase hypothesis. Moreover, in light of the H+-quenching of quinacrine fluorescence observed during ATP hydrolysis by vacuoles or tonoplast vesicles, the existence of a proton-pumping ATPase may be questioned.

Un nouveau colorant fluorescent pour la mesure du temps de relaxation brownienne des proteines

The conclusion is that the NBD‐chloride can be used, like the DNS‐chloride but is more reactive and stable and its fixation, on trypsin can be followed by measuring the absorbance of the dye at 470 nm.

Na/K competitive transport selectivity of (221) C lo-cryptand: effects of pH and carrier concentration

The kinetics of the competitive transport of Na+ and K+ ions across the membrane of large unilamellar vesicles (LUV) were determined when transport was induced by (221)C10-cryptand, an ionizable mobile carrier. The experiments were performed at various pH values (7.7 and 8.7) and carrier concentrations (0.1, 0.5 and 1.0 microM) in order to quantify the effects of these parameters on the Na/K competitive transport selectivity of this mobile carrier. At any given pH and carrier concentration, the apparent affinity of (221)C10 for Na+ was higher and less dependent on the concentration of the other competing ion than that for K+. The Na/K competitive transport selectivity (SC(Na/K)) of (221)C10 increased linearly with the Na+ concentrations, decreased hyperbolically with increasing those of K+ and was independent of the pH and of the carrier concentration. In equimolecular ionic mixtures, this competitive selectivity amounted to about 1.5 and when the pH rose, the carrier selectivity for Na+ over K+ ions was enhanced by cation competition compared to transport of cations as unique substrates. Equations were established to describe the variations of the competitive transport selectivity (SC) of cryptands, and for comparison of their noncompetitive selectivity (SNC), with the ionic concentrations, the Michaelis parameters of the cations and the pH. The reaction order in Na+ (n(Na)) increased significantly with decreasing the pH and the K+ concentration. The results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes.

High-performance liquid affinity chromatographic separations: Activation of matrices by 4,6-diphenylthieno[3,4-d]-1,3-dioxol-2-one-5,5-dioxide

Abstract High-performance liquid affinity chromatography requires activated matrices with specific properties. Standard matrices were modified and activated and then their characteristics were compared. A new activating method based on the use of 4,6-diphenylthieno[3,4- d ]-1,3-dioxol-2-one-5,5-dioxide was developed. The coupling of ligands with these new supports was very fast. The hydrolysis/aminolysis ratio was higher than with N-hydroxysuccinimide-activated matrices.

Dinoflagellate luminescence: Purification of a NAD(P)H-dependent reductase and of its substrate

The soluble enzymatic luminescent system of the dinoflagellate Pyrocystis lunula (luciferase-luciferin) is coupled with an enzymatic NAD(P)H-dependent reaction. The enzyme is a soluble reductase (Mr 47,000) which catalyzes, in the presence of NAD(P)H, the reduction of a molecule called P630. Reduced P630 has the same spectral characteristics as the purified luciferin. The luciferase can oxidize this reduced molecule with a light emission at 480 nm. These observations suggest that reduced P630 is a luciferin molecule. The oxidized form seems, in these conditions, to be the precursor of luciferin.

Transport of competing Na and K ions by (222) C10-cryptand, an ionizable mobile carrier: Effects of pH and temperature

The kinetics of the electroneutral exchange of competing sodium and potassium with protons across the membrane of large unilamellar vesicles (LUV) were determined at two pH values when transport was induced by the simultaneous presence of (222)C10-cryptand and FCCP (proton carrier) at various temperatures. The aim of the present work was to quantify the pH-dependent enthalpies of an ionizable mobile carrier affinities for competing alkali cations, and to focus on the effects of pH and temperature on the competitive transport selectivity of the carrier for K+ over Na+ ions. At any given temperature and pH, the apparent pH-dependent affinity of (222)C10 was higher for K+ than for Na+. The enthalpy of this affinity for K+ was significantly lower than that for Na+, whereas it varied similarly with the pH (delta H(KpHmK) = 32.8 and 37.0 kJ/mol, and delta H(KpHmNa) = 47.9 and 52.9 kJ/mol at pH 7.8 and 8.8, respectively). When using a kinetic model, the pH effect on these parameters was discriminated (delta H(KmK) = 37.9 kJ/mol and delta H(KmNa) = 53.9 kJ/mol). The pH-dependence of the delta H(KpHm) of the cations could therefore theoretically be shown to arise from the temperature-induced changes in the ionization of the buffer dissolved in the aqueous phases and of the amine groups of the binding cavity of the carrier. The K/Na competitive transport selectivity (Sc(K/Na)) of (222)C10 increased linearly with the K+ concentration. It decreased hyperbolically with increasing concentration of Na+ while being independent of pH at any given temperature. In equimolecular ionic mixtures, Sc(K/Na) varied from 2.2 to 3.0 when temperature rose from 20 degrees C to 35 degrees C (delta H(Sc(K/Na)) = 15.6 +/- 0.5 kJ/mol). The results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes.