Urease immobilized on aminated butyl acrylate-ethylenedimethacrylate copolymer

Urease was covalently immobilized on a new acrylic enzyme carrier: aminated butyl acrylate-ethylenedimethacrylate copolymer. The enzyme retained 56% of its original activity. The properties of the free and immobilized urease were determined and compared. The Michaelis constant was found to be higher for the immobilized urease than for the free one, while maximum reaction rate was lower for the immobilized enzyme. The stability of urease against change in pH was considerably improved by the immobilization. The immobilized urease showed very good storage stability and reusability. Resistance of the enzyme against heat inactivation at 70°C, however, was not found to be improved.     

Modification of jack bean urease thiols by thiosulphinates contained in garlic extract: DTNB titration studies

The reactivity of the thiol groups in urease with thiosulphinate contained in garlic extract was spectroscopically characterised. The enzyme was incubated with the garlic extract and the reaction progress curves were recorded in the presence of thiol-selective reagent 5,5′-dithiobis(2-nitrobenzoic acid). Simultaneously the enzyme residual activity was also determined. The process was studied in 50 mM phosphate buffer, at pH 7.8 and ambient temperature. It was found that thiosulphinates act as time- and concentration-dependent inactivators of urease. The observed decrease of the enzyme activity corresponds to the number of the urease thiols modified by thiosulphinates. The modification of half of all the urease thiols (18 of 36) causes a slight (only 8–10%) decrease of the catalytic activity. The modification of the remaining 18 thiols results in significant disturbance of urease action until complete loss of the catalytic function occurs. This provides the evidence that Cys⁵⁹², the critical residue for urease activity, belongs to the enzyme thiols, which are less reactive and more resistant to chemical modification than the other thiols.     

Urease immobilized on chitosan membrane: Preparation and properties

Urease was covalently immobilized on glutaraldehyde-pretreated chitosan membranes. The optimum immobilization conditions were determined with respect to glutaraldehyde pretreatment of membranes and to reaction of glutaraldehyde-pretreated membranes with urease. The immobilized enzyme retained 94% of its original activity. The properties of free and immobilized urease were compared. The Michaelis constant was about five times higher for immobilized urease than for the free enzyme, while the maximum reaction rate was lower for the immobilized enzyme. The stability of urease at low pH values was improved by immobilization; temperature stability was also improved. The optimum temperature was determined to be 65°C for the free urease and 75°C for the immobilized form. The immobilized enzyme had good storage and operational stability and good reusability, properties that offer potential for practical application.     

Inhibition of activity of urease in native form and immobilized on chitosan membrane by sodium fluoride

The influence of sodium fluoride inhibitor on the activity of urease in native form and immobilized covalently on glutaraldehyde-pretreated chitosan membrane was studied. Initially, in the presence of fluoride ions (I), a complex EI is formed which undergoes slow isomerization into a secondary form EI*. Both stages are represented on the progress curves. The dissociation constant of EI, K_i, for native and bonded urease were determined. The apparent rate constant as a function of the initial urea concentration for a given fluoride ion concentration was calculated, k = k ([I], [S]). It was found that sodium fluoride is a competitive slow-binding inhibitor of urease. Hydrolysis of urea in the standard conditions, at pH = 7.0, at 25° and a constant ionic strength and variable concentrations of substrate and of inhibitor has been studied. The reaction was initiated by addition of enzyme and was observed over a period of 10 min. The rate of hydrolysis at any point of time for both forms of urease, free and bound, may be calculated from the inhibition constants K_i, K*_i and apparent rate constant, k. It was found that immobilized urease is more resistant to the action of the inhibitor than the native one. This property offers potential for practical application.     

Competitive inhibitors of free and chitosan-immobilized urease

Renal proximal tubules isolated from the rat possess nitric oxide synthase (NOS) activity that is calcium/calmodulin dependent and stereoselectively inhibited by NG-monomethyl-arginine (NMMA). In the absence of added Ca2+ and calmodulin, activity was reduced 84 +/- 13% compared with the activity in the presence of 2 mM Ca2+ and 25 micrograms/mL calmodulin. Inhibition by EGTA (10 mM) was 95 +/- 5% and by calmidazolium (R24571, 250 microM) was 99 +/- 1%. Inhibition by L-NMMA (100 microM) was 78 +/- 13% and by D-NMMA (100 microM) was 7 +/- 7%. The majority of NOS activity was found in the soluble fraction. NOS activity in isolated proximal tubules was also examined 6 hr after a single i.v. injection of lipopolysaccharide. Activity was increased significantly (P < 0.05) in the soluble fraction by 2-fold [from 0.320 +/- 0.052 to 0.648 +/- 0.046 (nmol/mg protein/30 min)] and in the particulate fraction by 3-fold [from 0.081 +/- 0.030 to 0.256 +/- 0.034 (nmol/mg protein/30 min)]. All activities were inhibited by EGTA. These data demonstrate that proximal tubules express a calcium/calmodulin-dependent NOS activity that is increased in vivo by lipopolysaccharide.