Analysis of progress curves for a highly concentrated Michaelian enzyme in the presence or absence of product inhibition.

Methods are given for analysing the time course of an enzyme-catalysed reaction when the concentration of the enzyme itself is high, a situation which is often found in vivo. (1) The integrated form of the kinetic equation for a concentrated Michaelian enzyme in absence of product inhibition is given. Parameters are shown to be calculated easily using non-linear fitting procedures. (2) A general algorithm to analyse progress-curve data in more complex cases (i.e. when the analytical form of the integrated rate equation is not known or is exceedingly complex) is proposed. This algorithm may be used for any enzyme mechanism for which the differential form of the kinetic equation may be written analytically. We show that the method allows differentiation between the main types of product inhibition which may occur in the case of a highly concentrated Michaelian enzyme.     

NRP/Optineurin Cooperates with TAX1BP1 to Potentiate the Activation of NF-κB by Human T-Lymphotropic Virus Type 1 Tax Protein

Nuclear factor (NF)-κB is a major survival pathway engaged by the Human T-Lymphotropic Virus type 1 (HTLV-1) Tax protein. Tax1 activation of NF-κB occurs predominantly in the cytoplasm, where Tax1 binds NF-κB Essential Modulator (NEMO/IKKγ) and triggers the activation of IκB kinases. Several independent studies have shown that Tax1-mediated NF-κB activation is dependent on Tax1 ubiquitination. Here, we identify by co-immunoprecipitation assays NEMO-Related Protein (NRP/Optineurin) as a binding partner for Tax1 in HTLV-1 infected and Tax1/NRP co-expressing cells. Immunofluorescence studies reveal that Tax1, NRP and NEMO colocalize in Golgi-associated structures. The interaction between Tax1 and NRP requires the ubiquitin-binding activity of NRP and the ubiquitination sites of Tax1. In addition, we observe that NRP increases the ubiquitination of Tax1 along with Tax1-dependent NF-κB signaling. Surprisingly, we find that in addition to Tax1, NRP interacts cooperatively with the Tax1 binding protein TAX1BP1, and that NRP and TAX1BP1 cooperate to modulate Tax1 ubiquitination and NF-κB activation. Our data strongly suggest for the first time that NRP is a critical adaptor that regulates the assembly of TAX1BP1 and post-translationally modified forms of Tax1, leading to sustained NF-κB activation.     

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.     

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.     

Mössbauer spectroscopy study of iron overloaded livers

Absorption 57Fe M?ssbauer spectra have been carried out directly on fresh or lyophylized tissues of liver with either normal iron depot or iron overload. Two types of overloading have been studied: primary iron overload due to an excessive intestinal iron absorption and secondary iron overload (hemosiderosis) produced in beta-thalassemia patients by hypertransfusional therapeutics. The M?ssbauer spectra, at room temperature, 77 and 4.2 K, on normal liver samples, are typical for the ferritin-hemosiderin compounds. In the spectra, performed on hemosiderosis liver samples, there appears, in addition to ferritin and hemosiderin, a new iron molecular environment, typical of high spin ferric iron and characterized by a superparamagnetic behaviour which begins at high temperature (above 77 K). This new component does not show up in the primary iron overload cases and seems characteristic of the physiological process which induces the iron overload.