Interactions between verapamil and neutral and acidic liposomes: effects of the ionic strength.

Patients with cancer often develop major electrolyte disorders, which are aggravated by radiation therapy and chemotherapy and by the concomitant impairment of the renal function and the development of drug resistance. In addition, tumour cells have membranes with more negative charges than normal eukaryotic cells. This study was designed to test the hypothesis that the ability of the Ca(2+) blocker verapamil to mediate the reversal of multidrug resistance (MDR) by interacting with the membrane phospholipids may be correlated with the ionic strength and membrane surface potential in resistant tumours. The permeation properties of verapamil, which is the best-known MDR-modulator, were therefore studied by quantifying its ability to induce the leakage of carboxyfluorescein through unilamellar liposomes containing various mole fractions of phosphatidic acid (x(EPA)=0, 0.1 and 0.3), at four different ionic strengths (I=0.052, 0.124, 0.204 and 0.318 M). The dye leakage induced by verapamil varied greatly with I, depending on x(EPA). The permeation process was a co-operative one (1.3

Na/K competitive transport selectivity of (221) C10-cryptand: effect of temperature.

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 at various temperatures in order to quantify the temperature-dependence of the Na/K competitive transport selectivity of this ionizable mobile carrier. At any given temperature, 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+. Its enthalpy for Na+ (delta H(KmNa) = 50.6 kJ/mol) was not significantly different from that for K+ (delta H(KmK) = 52.7 kJ/mol). The Na/K competitive transport selectivity (SC(Na/K)) of (221)C10 increased linearly with the Na+ concentrations and decreased hyperbolically with increasing those of K+. When the cation concentrations were equal, this competitive selectivity amounted to about 2 at any given temperature. 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 and the Michaelis parameters of the cations. It is theoretically demonstrated that the ratio between the competitive and noncompetitive transport selectivities, i.e., SC/SNC, of mobile carriers does not depend on the Jmax of the competing ions and that its value amounts to 1 when the specific concentrations (C'S/Km) of the ions are equal. Under these conditions, the transport selectivity of any given mobile carrier has the same value whether determined from competition or separated experiments. The reaction order in Na+ (n(Na)) increased significantly as the temperature rose and decreased significantly as the K+ concentration increased. The results are discussed in terms of the structural, physicochemical and electrical characteristics of carriers and complexes.

Na/K competitive transport selectivity of (221)C10-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.

Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli.

Glucosamine-6-phosphate isomerase (deaminase), (2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) has been purified to homogeneity from Escherichia coli B as judged by several criteria of purity. The procedure included ammonium sulfate fractionation, anion-exchange chromatography and a biospecific affinity chromatography step with N-epsilon-amino-n- caproyl -D-glucosamine 6-phosphate bound to agarose as the ligand, the elution being performed with GlcNAc6 P. The enzyme appears to be an hexamer of about 178 kDa, composed of six subunits of 29 700 +/- 300 Da; the isoelectric point was 6.0-6.1 and the sedimentation constant 9.0 S. The amino-acid composition of the enzyme was determined and a value for E1%275 of 4.55 was calculated. The molecular activity was 1800 s-1 for the deamination reaction and 455 s-1 for the reaction of GlcN6 P formation. A positive homotropic cooperativity was found for both sugar substrates; it was stronger for GlcN6 P in the deamination reaction (Hill number 2.7 at pH 7.7). Ammonia behaved as a Michaelian substrate. Cooperativity was abolished by 0.1 mM GlcNAc6 P; this allosteric modulator activated the reaction in both directions, with a positive K-effect upon both sugar phosphates, but had no effect on Km for ammonia. The initial velocity patterns for the amination reaction were obtained under conditions of hyperbolic kinetics produced by GlcNAc6 P; the Km values for the allosteric substrates were determined under the same conditions, and their dependence upon pH was studied.

Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

A circularly permuted (cp) variant of the phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been constructed with N- and C-termini created within the coenzyme binding domain. The cp variant has a kcat value equal to 40% of the wild-type value, whereas Km and KD values for NAD show a threefold decrease compared to wild type. These results indicate that the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permuted variant and that NAD binding is tighter. Reversible denaturation experiments show that the stability of the variant is only reduced by 0.7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two structural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.

Dynamics aspects of long distance functional interactions between membrane-bound enzymes.

The aim of this mini review is to study how an organized charged milieu, such as a membrane, may alter functional long-distance interactions between bound enzymes. Two questions are more specifically considered. The first is to know whether the overall response of a bound enzyme is dependent upon the degree of spatial order of fixed charges and enzymes molecules. The second is to determine whether electric interaction between the fixed charges of the matrix and the charged substrate may generate hysteresis loop of substrate concentration as well as oscillations of this concentration at the surface of the membranes. These effects that have been shown to occur at the surface of membranes, are not the result of intrinsic properties of enzymes. They appear as the consequence of the interplay between functional long-distance interactions between bound enzyme systems and electric repulsion effects of mobile ions. They may be viewed as supramolecular devices that allow storing information from the external milieu.

Spatial order as a source of kinetic cooperativity in organized bound enzyme systems.

When enzyme molecules are distributed within a negatively charged matrix, the kinetics of the conversion of a negatively charged substrate into a product depends on the organization of fixed charges and bound enzyme molecules. Organization is taken to mean the existence of macroscopic heterogeneity in the distribution of fixed charge density, or of bound enzyme density, or of both. The degree of organization is quantitatively expressed by the monovariate moments of charge and enzyme distributions as well as by the bivariate moments of these two distributions. The overall reaction rate of the bound enzyme system may be expressed in terms of the monovariate moments of the charge density and of the bivariate moments of charge and enzyme densities. The monovariate moments of enzyme density do not affect the reaction rate. With respect to the situation where the fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by these two types of moments, generates an increase or decrease of the overall reaction rate as well as a cooperativity of the kinetic response of the system. Thus both the alteration of the rate and the modulation of cooperativity are the consequence of a spatial organization of charges with respect to the enzyme molecules. The rate equations have been derived for different types of organization of fixed charges and enzyme molecules, namely, clustered charges and homogeneously distributed enzyme molecules, clustered enzyme molecules and homogeneously distributed charges, clusters of charges and clusters of enzymes that partly overlap, and clusters of enzymes and clusters of charges that are exactly superimposed. Computer simulations of these equations show how spatial molecular organization may modulate the overall reaction rate.

pHmetrical determination of the glucosamine-6-phosphate isomerase deaminase reverse reaction.

In the reverse direction, the reaction catalyzed by glucosamine 6-phosphate isomerase deaminase consumes ammonia and forms GlcN6P. As a consequence of the formation of a product with a lower pK than the substrates, a measurable pH drop in the reaction medium is produced. This property can be used to follow potentiometrically the course of the reaction. This property can be used to follow potentiometrically the course of the reaction. The usefulness of the method is demonstrated obtaining the inhibition pattern by GlcN6P when Fru6P is the varied substrate.

Sulfhydryl groups of glucosamine-6-phosphate isomerase deaminase from Escherichia coli.

Glucosamine-6-phosphate isomerase deaminase (2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) from Escherichia coli is an hexameric homopolymer that contains five half-cystines per chain. The reaction of the native enzyme with 5',5'-dithiobis-(2-nitrobenzoate) or methyl iodide revealed two reactive SH groups per subunit, whereas a third one reacted only in the presence of denaturants. Two more sulfhydryls appeared when denatured enzyme was treated with dithiothreitol, suggesting the presence of one disulfide bridge per chain. The enzyme having the exposed and reactive SH groups blocked with 5'-thio-2-nitrobenzoate groups was inactive, but the corresponding alkylated derivative was active and retained its homotropic cooperativity toward the substrate, D-glucosamine 6-phosphate, and the allosteric activation by N-acetyl-D-glucosamine 6-phosphate. Studies of SH reactivity in the presence of enzyme ligands showed that a change in the availability of these groups accompanies the allosteric conformational transition. The results obtained show that sulfhydryls are not essential for catalysis or allosteric behavior of glucosamine-6-phosphate deaminase.

Structural and functional properties of a multi-enzyme complex from spinach chloroplasts. 2. Modulation of the kinetic properties of enzymes in the aggregated state.

The carboxylase activity of free ribulose 1,5-bisphosphate carboxylase-oxygenase has been compared to that of the five-enzyme complex present in chloroplasts. Kinetic results have shown that the V/active site is lower for the free enzyme than for the complex. Conversely the Km is smaller for the complex than for the free enzyme. This implies that the catalytic activity of the enzyme is enhanced when it is embedded in the complex. Under reducing conditions and in the presence of reduced thioredoxin, inactive oxidized phosphoribulokinase, free in solution or inserted in the multi-enzyme complex, becomes active. The kinetics of this activation process has been studied and shown to be exponential. The time constant of this exponential decreases, for the free enzyme, as thioredoxin concentration is increased. Alternatively, for the enzyme embedded in the complex, this time constant increases with thioredoxin concentration almost in a linear fashion. This implies that the complex is much more rapidly activated by reduced thioredoxin than is the free phosphoribulokinase. The variation of the amplitude of this activation process as a function of thioredoxin concentration is a hyperbola. The concentration of thioredoxin which results in half the asymptotic value of this hyperbola is smaller for the complex than for the free enzyme. A kinetic model has been proposed and the dynamic equations resulting from this model have been derived. They fit the experimental results exactly. From the variation of the amplitude of the activation process one may derive the binding constants of thioredoxin on either the oxidized enzyme or on a partly dithiothreitol-reduced enzyme (both of them free or inserted in the complex). In either case, the affinity of reduced thioredoxin is larger for the complex than for the free enzyme. The individual values of some of the rate constants have also been estimated from the variation of the time constants as a function of thioredoxin concentration. Taken together, these results show that at least two enzymes, ribulose 1,5-bisphosphate carboxylase-oxygenase and phosphoribulokinase, have quite different kinetic properties depending on whether they are in free solution or embedded in the multi-enzyme complex.

Biological activities and structural properties of the atypical bacteriocins mesenterocin 52b and leucocin b-ta33a.

The antibacterial spectra and modes of action of synthetic peptides corresponding to mesenterocin 52B and leucocin B-TA33a greatly differ despite their high sequence homology. Circular dichroism experiments establish the capacity of each of these two peptides to partly fold into an amphiphilic helix that might be crucial for their adsorption at lipophilic-hydrophilic interfaces.

Molecular organization and clustering of cell-wall-bound enzymes as a source of kinetic apparent co-operativity.

When fixed charges and enzyme molecules are not homogeneously distributed in a matrix, the degree of organization of charges, of enzyme molecules and of charges with respect to enzyme molecules modulate the enzyme reaction rate. The overall reaction velocity of the bound enzyme system may be expressed in terms of monovariate moments of the charge density distribution and of the bivariate moments of the charge and enzyme density distributions. With respect to the situation where fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by the monovariate and bivariate moments results in an increase or a decrease, of the overall reaction rate, as well as in the appearance of a kinetic cooperativity. The degree of spatial organization of objects may be expressed quantitatively through the concept of minimal spanning tree. This concept may thus be applied to the quantification of the degree of order that may exist in the bidimensional distribution of enzyme molecules in a charged matrix. Primary walls of isolated plant cells in sterile culture behave as a polyanion and contain different enzymes. The spatial distribution in sycamore cell walls of an acid phosphatase has been studied through the concept of minimal spanning tree and shown to be non-randomly distributed in the polyanionic matrix, but clustered in that matrix. This spatial organization results in a modulation of the reaction rate of the cell-wall-bound phosphatase reaction. Both the theoretical and experimental results presented in this study leave little doubt as to the validity of the idea that in situ the organization of fixed charges and enzyme molecules modulate the overall dynamics of enzyme reactions.