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The aim of this study was to define a new indicator for the quality of wastewaters that are released into the environment. A quality index is proposed for wastewater samples in terms of the inertness of wastewater samples toward enzyme activity. This involves taking advantage of the sensitivity of enzymes to pollutants that may be present in the waste samples. The effect of wastewater samples on the rate of a number of different enzyme-catalyzed reactions was measured, and the results for all the selected enzymes were analyzed in an integrated fashion (multi-enzymatic sensor). This approach enabled us to define an overall quality index, the "Impact on Enzyme Function" (IEF-index), which is composed of three indicators: i) the Synoptic parameter, related to the average effect of the waste sample on each component of the enzymatic sensor; ii) the Peak parameter, related to the maximum effect observed among all the effects exerted by the sample on the sensor components; and, iii) the Interference parameter, related to the number of sensor components that are affected less than a fixed threshold value. A number of water based samples including public potable tap water, fluids from urban sewage systems, wastewater disposal from leather, paper and dye industries were analyzed and the IEF-index was then determined. Although the IEF-index cannot discriminate between different types of wastewater samples, it could be a useful parameter in monitoring the improvement of the quality of a specific sample. However, by analyzing an adequate number of waste samples of the same type, even from different local contexts, the profile of the impact of each component of the multi-enzymatic sensor could be typical for specific types of waste. The IEF-index is proposed as a supplementary qualification score for wastewaters, in addition to the certification of the waste's conformity to legal requirements., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In order to define the ability of bovine lens aldose reductase (ALR2) to generate polyols from aldoses, the quantitative determination of glycerol in the presence of glyceraldehyde was performed by gas chromatography after derivatization with trifluoroacetic anhydride. The proposed method appears to be useful in quantifying low amounts of glycerol in the presence of relatively high concentrations of glyceraldehyde and in following glycerol formation in enzyme assay conditions. The generation of one equivalent of glycerol in the presence of ALR2, is paralleled by the oxidation of one equivalent of NADPH. A similar result was obtained when S-glutathionyl-modified ALR2 was used, instead of the native enzyme, as a catalyst of glyceraldehyde reduction. Sorbinil, a classical ALR2 inhibitor, present in the enzyme assay mixture, inhibits to the same extent both NADPH oxidation and glycerol formation. The demonstration of the stoichiometric ratio of 1:1 occurring in the presence of bovine lens ALR2 between the synthesis of glycerol from D, L -glyceraldehyde and the oxidation of NADPH, rules out doubts concerning the ability of the enzyme to catalyse the reduction of aldoses to the corresponding polyalcohols. Possible autooxidation processes of glyceraldehyde, in the enzyme assay conditions, appear to be irrelevant with respect to the enzyme-catalysed reduction of the aldose. This would indicate that the spectrophotometric monitoring of NADPH oxidation at 340 nm, in the presence of ALR2, is a reliable method to assay the enzyme activity., (Copyright 2000 Academic Press.)

The reversibility of S-thiolation of aldose reductase was shown in intact bovine lens subjected to oxidative stress. The glutathione modified aldose reductase generated in the lens as a consequence of hyperbaric oxygen treatment was recovered in its reduced form following culturing in normobaric air conditions. Nucleus and cortex were differently affected by both oxidative treatment and normobaric air recovery. The extent of S-thiolation of aldose reductase appeared to be higher in the nucleus than in the cortex. Moreover, the nucleus, but not the cortex, was unable to completely recover from the protein S-thiolation process. The ratios of GSH/GSSG and NADPH/NADP(+)as well as the Energy Charge values were determined in the cortex and nucleus both after oxidative stress and recovery. The results are consistent with the existence of a quite well-defined boundary between the two lens regions. Moreover, they are supportive of the hypothesis that thiol/disulfide exchange has the potential to be a regulatory mechanism for certain enzymes which can modulate the flux of NADPH inside the cell., (Copyright 2000 Academic Press.)

Aldose reductase inhibition is one of the therapeutic strategies that has been proposed to prevent or ameliorate long term diabetic complications including retinopathy and sugar cataract. Rats were fed with a galactose rich diet and the aldose reductase inhibitor Tolrestat was topically delivered by ocular instillation. The levels of lens aldose reductase activity, galactitol and the onset of cataract were evaluated during and after treatment with the inhibitor. Topical application of 1-3% Tolrestat (10 microl) four times daily resulted, after 9 days, in a significant decrease in the enzyme activity. Well after interrupting treatment with the drug, the enzyme activity remained impaired and galactose induced cataract was prevented. Our findings may represent the basis for therapeutic plans to prevent sugar cataract by long term cyclic treatments with aldose reductase inhibitors, with reduction in drug doses and side effects., (Copyright 1999 Academic Press.)

Bovine lens aldose reductase (ALR2) is readily modified by glutathione disulphide (GSSG) to an enzyme form (GS-ALR2) exhibiting a reduced catalytic efficiency with all the substrates tested and a reduced susceptibility to inhibition. The modification, which is completely reversed by reduced glutathione (GSH) or dithiothreitol occurs by a pseudo-first-order process with respect to the enzyme and a second order rate constant of 30 +/- 0.1 mol-1 min-1 at 25 degrees C was determined. By measuring the residual activity of ALR2 incubated in different glutathione redox buffers at 25 degrees C, an apparent redox equilibrium constant of 1.4 +/- 0.1 was evaluated. Thus the rate and the maximal extent of ALR2 inactivation are proportional to the redox ratio of the thiol used as modifying agent (i.e. [GSH]/[GSSG]). The stoichiometric reversibility of the enzyme modification might be impaired by a reduced solubility of GS-ALR2 with respect to ALR2 and by an increased susceptibility of the modified enzyme to proteolysis. While the native enzyme form is rather insensitive to proteolytic breakdown. GS-ALR2 is easily degraded by chymotrypsin with the generation of a peptide of 26 kDa with an aminoacid sequence at the aminoterminal side compatible with proteolysis at level of Tyr 7 of aldose reductase. A reduced efficiency in the enzyme-cofactor binding following the GSSG dependent modification of ALR2, appears to be associated to the thiol accessibility of GS-ALR2 measured at different temperatures. GS-ALR2 is characterized by the presence of one glutathione residue, linked through a mixed disulphide bond. This is sustained by: (i) the isoelectric point for the modified enzyme of 4.75, which is 0.1 pH units lower than that observed for the native enzyme, which indicates the contribution of an acidic residue to the pI of GS-ALR2; (ii) the incorporation of radioactivity coming from [3H] labelled GSSG accounting for the presence of one equivalent of glutathione per mole of enzyme. Besides being a general feature of protein reactivity in oxidative conditions, the glutathione-mediated ALR2 modification might be part of a cell strategy to preserve reducing power in conditions of oxidative stress.