Roger Giordani

Enzymatic and physiological properties of the tungsten‐substituted molybdenum TMAO reductase from Escherichia coli

The trimethylamine N‐oxide (TMAO) reductase of Escherichia coli is a molybdoenzyme that catalyses the reduction of the TMAO to trimethylamine (TMA) with a redox potential of + 130 mV. We have successfully substituted the molybdenum with tungsten and obtained an active tungsto‐TMAO reductase. Kinetic studies revealed that the catalytic efficiency of the tungsto‐substituted TMAO reductase (W‐TorA) was increased significantly (twofold), although a decrease of about 50% in its kcat was found compared with the molybdo‐TMAO reductase (Mo‐TorA). W‐TorA is more sensitive to high pH, is less sensitive to high NaCl concentration and is more heat resistant than Mo‐TorA. Most importantly, the W‐TorA becomes capable of reducing sulphoxides and supports the anaerobic growth of a bacterial host on these substrates. The evolutionary implication and mechanistic significance of the tungsten substitution are discussed.

Tributyroylglycerol hydrolase activity in Carica papaya and other latices

Abstract Lipase activity (measured with tributyrin as substrate) was detected for the first time in fresh latex of branched non-articulated laticifers of Euphorbia characias, Asclepias curassavica and A. serilofera. Activity was also detected in spray-dried latex from anastomosing articulated laticifers of Carica papaya. The lipolytic activity of all the latices was associated with sedimentable particles. Attempts to solubilize the enzymatic activity from particulate fractions of Carica papya latex were unsuccessful. The esterase inhibitors phenylmethylsulphonyl fluoride or benzamidine had no effect on the lipase activity. The enzyme in Carica papya latex shows greatest activity with tributyrin and is stable in non-water-miscible organic solvents.

Characterization of the plasma membrane ATPase of Candida tropicalis

1) Plasma membrane vesicles from Candida tropicalis were isolated from protoplasts by differential centrifugation and purified in a continuous sucrose gradient. 2) The plasma membrane bound ATPase was characterized. It is highly specific for ATP and requires Mg2+. It is stimulated by K+, Na+ and NH4+. Lineweaver-Burk plots for ATPase activity are linear with a Vmax of 4.2 mumoles of ATP hydrolyzed min-1.mg-1 protein and a Km for ATP of 0.76 mM. The ATPase activity is inhibited competitively by ADP with a Ki of 1.7 mM and non competitively by vanadate with a Ki of 3 microM. The activity is unaffected by oligomycin or azide but is sensitive to DCCD.

Purification and properties of N-acetyl-β-d-glucosaminidase from Hevea brasiliensis latex

Abstract β-N-Acetylglucosaminidase from Hevea brasiliensis latex was isolated by anion exchange and gel filtration chromatography. The purified enzyme showed a single protein band on native electrophoresis. It is a glycoprotein with a molecular mass of 92 kDa, consisting of two 46-kDa subunits and has a glycosidic content of 16%. It shows optimal activity at pH 6.0 and at 50°C; its amino acid composition has been established. It is inhibited by several monovalent or divalent ions. The enzyme hydrolyses p- nitrophenyl -N- acetyl -β- d - glucosaminide (pNP-β- d -GlcNAc) with apparent Km and Vm values of 1.13 mM and 185 mM min−1 mg−1 of protein, respectively, at the optimum pH. p- Nitrophenyl -N- acetyl -β- d - galactosaminide (pNP-β- d -GalNAc) can also be used as a substrate but is less efficient. Glucosamine and glactosamine were competitive inhibitors with Ki values of 2.2 mM and 7.5 mM, respectively, at pH 6.0. The results of kinetic studies suggest that two ionizable groups with pK values of about 4 and 6.5 may take part in the reaction, possibly in the substrate binding. The vacuolar location of the enzyme is in agreement with the idea that the latex might play a lysosomal role in interacellular digestion processes.

Kinetic Studies of a Soluble αβ Complex of Nitrate Reductase A from Escherichia Coli

A soluble alpha beta complex of nitrate reductase can be obtained from a strain of Escherichia coli that lacks the narI gene and expresses only the alpha and beta subunits. The beta subunit contains four Fe-S centres and the alpha subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced. Despite the lack of the gamma subunit of the complete enzyme, this complex can still catalyse the reduction of nitrate with artificial electron donors such as benzyl viologen, so that it is suitable for studying the transfer of electrons between these two types of redox centre. To examine whether the electrons from reduced benzyl viologen are initially delivered to the Fe-S centres, or directly to the molybdenum cofactor, or both, we have studied the steady-state kinetics and the binding of benzyl viologen to the alpha beta complex and mutants alpha beta* with altered beta subunits. Reduction of the enzyme by reduced benzyl viologen in the absence of nitrate showed that all four Fe-S centres and the molybdenum cofactor could be reduced. Two classes of site with different equilibrium constants could be distinguished. The kinetic results suggest that benzyl viologen supplies its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on the square of the concentration of the electron donor. A reaction mechanism is proposed for the reduction of nitrate catalysed by the alpha beta complex of nitrate reductase with artificial electron donors.

Elution of acid phosphatase from sycamore cell walls

Abstract In isolated cell wall fragments of cultured sycamore cells, acid phosphatase activity was preferentially located on one side. This side has been identified as the external side of the cell wall. About 60% of acid phosphatase activity could be solubilized from cell wall fragments. Electron micrographs showed that the majority of the solubilized enzyme came from the external area of the wall.

Action of Carica papaya latex on cell wall glycosidases from Lactuca sativa pith

Abstract The various glycosidase activities from Carica papaya latex and pith cell walls from Lactuca sativa were measured by using pNP-glycoside substrates. A decrease of enzymic activities was obtained by incubation of isolated cell walls with C. papaya latex. Thus parietal activities decreased by the following amounts: N -acetyl-β- d -glucosaminidase by 83%, β- d -glucosidase by 45%, β- d -fucosidase by 53% and α- d -galactosidase by 25% after an incubation of 24 hr. These results indicate that C. papaya latex has no stimulatory effect on the cell wall enzymes tested. Addition of cysteine protease inhibitor to medium culture causes a smaller decrease of parietal glycosidic activities. Carica papaya latex proteases and glycosidases are, therefore, responsible for the decrease of cell wall glycosidic activities after action of this latex. A comparison with the increase of glycosidic activities of Candida albicans cultured in a medium supplemented with latex indicates a possible stimulation of glycoprotein synthesis by Golgi apparatus for this enzymic activation.

Purification and molecular properties of an acid phosphatase from Asclepias curassavica latex

Abstract An acid phosphatase was isolated and purified from Asclepias curassavica latex. This phosphatase is the first to be obtained in the pure state from non-articulated laticifers. The enzyme has a molecular weight close to 27 000 and its amino acid composition was determined. It catalyses the hydrolysis of various phosphorylated compounds, p- nitrophenyl phosphate being the most efficient as found for most other phosphatases. The optimal pH was found to be approx. 6.0 and at this pH the Michaelis constant was 0.69 mM. Phosphate was a competitive inhibitor indicating that it is the last product to be liberated during the reaction course. Pyrophosphate, arsenate, molybdate, cupric and mercuric salts were inhibitory. Calcium, magnesium, potassium and tungsten had no effect. The physiological significance of the presence of this enzyme in the latex was discussed.

An investigation into the feasibility of using azide-insensitive ATPase and ConA as yeast plasma membrane markers.

Cytochemical localization of Concanavalin A binding sites in protoplasts of Candida tropicalis, investigated with glycosylated-ferritin and electron microscopy, showed that the lectin was specifically bound to the external protoplast surface. Thus, the plasma membranes have been labelled with 125I-Concanavalin A and followed through the isolation procedure. Relative distribution of 125I-radioactivity and azide-insensitive ATPase activity in the obtained fractions, suggested that this enzyme was an equivocal plasma membrane marker. Despite the presence of internal Concanavalin A binding sites, Concanavalin A could be used unambiguously as an exogenous plasma membrane marker of intact protoplasts.

Stimulation of lipase and N-acetyl-β-d-glucosaminidase activities in Euphorbia characias latex by contact of free fatty acids with roots

Abstract A transitory increase in the basal levels of lipase and N- acetyl-β- d -glucosaminidase activity was observed in the latex of Euphorbia characias plants with their roots in contact with the free fatty acids, butyric, 3-butenoic and caprylic. Incorporation of [14C-1] -butyric acid into latex was also demonstrated.