Alain Dupaix

Inactivation of α-chymotrypsin by a bifunctional reagent, 2-bromomethyl-3,1-benzoxazin-4-one

Abstract α-Chymotrypsin is selectively and irreversibly inactivated by 2-bromomethyl-3, i -benzoxazin-4-one (Compd Ib) at neutral pH. Reaction of the enzyme with this activated ester leads rapidly to a relatively stable acyl-enzyme in which intramolecular alkylation of a single methionine residue (likely methionine-192) followed by hydrolysis of the acyl-enzyme bond occurs. Kinetic evidence for such a process is found in the measurements of proflavin displacement accompanying the reaction. The irreversibly modified enzyme still possesses its intact active site but its activity towards specific substrates is altered. Alkylation of the enzyme increases mainly K m but does not change appreciably k cat . The inhibition constants of specific inhibitors such as proflavin or indole are increased several times. From crystallographic data it is known that methionine-192 forms the lid of the specificity cavity in the enzyme. Therefore a bulky substituent such as the 2-acetamido benzoic acid on the sulfur atom of this methionine might sterically hinder the substrate binding by blocking the approach of the binding site.

ATP synthesis and pyrophosphate-driven proton transport in tonoplast-enriched vesicles isolated from Catharanthus roseus

In the presence of PPi as an energy source, the tonoplast‐bound inorganic pyrophosphatase from Catharanthus roseus cells is able to create a proton‐gradient which can drive the synthesis of ATP from ADP and Pi. ATP synthesis is linked to the pH‐gradient dissipation as monitored by the recovery of the fluorescence intensity of quinacrine and by the amount of synthesized ATP measured by the bioluminescent luciferin/luciferase assay. Proton gradient and ATP synthesis were suppressed by the protonic ionophore gramicidin D.

Separation of polar, steric and specific effects in the α-chymotrypsin-catalyzed hydrolysis of acyl-substituted p-nitrophenyl esters

Abstract The α-chymotrypsin-catalyzed hydrolysis of various acyl-substituted p-nitrophenyl esters has been studied at 25°C, between pH 6 and 8. The substituent effects on the deacylation rates of corresponding acyl-chymotrypsins have been analyzed using the Taft-Ingold relationship and contributions of polar, steric and specific effects, separated. A specificity constant S has been defined and its value discussed in relation to the structure of the substrate.

Design of a chemiluminescent and bioluminescent photometer.

An apparatus for chemi- and bioluminescence measurements is described. The main features are in high sensitivity: for example, 5 10(-12) M adenosine triphosphate, 5 10(-7) M glucose, 2 10(-9) M perhydrol and 4 10(-13) M peroxidase are easily detected; its fast mixing time and the possibility of injecting a reactant, at any time, enabling kinetic studies. Moreover, its compactness and 12 V battery supply allow measurements outside of laboratories.

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.

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.

The influence of temperature on the α-chymotrypsin-catalyzed hydrolysis of N-acylamino acid p-nitrophenyl esters*

Summary The effect of temperature on the deacylation step in the α-chymotrypsin-catalyzed hydrolysis of several N-acyl-L (or D)- amino acid p-nitrophenyl esters has been studied. A compensation in the activation parameters ΔH ‡ and ΔS ‡ for this step has been found. This phenomenon is discussed in terms of the breaking or formation of specific interactions between the substrate and the enzyme, in the transition state of the deacylation reaction.

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.

Characterization of tonoplast-enriched vesicles isolated by gradient density-fractionation of suspension-cultured cells

A tonoplast-enriched microsomal fraction was isolated from Catharanthus roseus cells. It was characterized by structural and functional criteria. This fraction presented a homogeneous size distribution as shown by quasi-elastic light scattering and a homogeneous density on self-generated gradients of Percoll. The mean diameter of the vesicles was estimated to be 0.30 micron and the buoyant density around 1.04 g/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of its polypeptide pattern was in good agreement with the one obtained for tonoplast purified from isolated vacuoles. According to enzymatic assays and inhibition tests, this fraction possessed pyrophosphate and ATP-dependent proton pumps and very low contamination by submitochondrial particles, endoplasmic reticula and Golgi membranes. In light of our previous published results on tonoplast purified from isolated vacuoles, the very low extent of AMP hydrolysis by the microsomal fraction is interpreted as supplementary proof in favor of a tonoplast-enriched fraction.

THE FLUORESCENT PRODUCT OF SCALEWORM BIOLUMINESCENT REACTION: AN IN VITRO STUDY

Abstract— The fluorescence of scaleworms has been attributed by Harvey (1952) to a product of the bioluminescent reaction confined in the scales of these worms. We have purified this fluorescent molecule by gel filtration. This compound has an apparent low molecular weight as shown by polyacrylamide gel clectrophoresis in the presence of SDS. The yield of the fluorescent product, after gel filtration, varies with the stimulation of the bioluminescence, triggered either chemically or enzymatically. The fluorescence spectrum of the purified product is similar to the one observed in vivo, with a maximum centered at 520 nm. Consequently, the fluorescent compound isolated is likely the reaction product of the bioluminescent reaction.