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The ability to catalyse a reaction acting on different substrates, known as “broad-specificity” or “multi-specificity”, and to catalyse different reactions at the same active site (“promiscuity”) are common features among the enzymes. These properties appear to go against the concept of extreme specificity of the catalytic action of enzymes and have been re-evaluated in terms of evolution and metabolic adaptation. This paper examines the potential usefulness of a differential inhibitory action in the study of the susceptibility to inhibition of multi-specific or promiscuous enzymes acting on different substrates. Aldose reductase is a multi-specific enzyme that catalyses the reduction of both aldoses and hydrophobic cytotoxic aldehydes and is used here as a concrete case to deal with the differential inhibition approach.

Cytosolic 5′-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.

The hyperactivity of aldose reductase (AR) on glucose in diabetic conditions or on glutathionyl-hydroxynonenal in oxidative stress conditions, the source of cell damage and inflammation, appear to be balanced by the detoxifying action exerted by the enzyme. This detoxification acts on cytotoxic hydrophobic aldehydes deriving from membrane peroxidative processes. This may contribute to the failure in drug development for humans to favorably intervene in diabetic complications and inflammation, despite the specificity and high efficiency of several available aldose reductase inhibitors. This paper presents additional features to a previously proposed approach, on inhibiting the enzyme through molecules able to preferentially inhibit the enzyme depending on the substrate the enzyme is working on. These differential inhibitors (ARDIs) should act on glucose reduction catalyzed by AR without little or no effect on the reduction of alkenals or alkanals. The reasons why AR may be an eligible enzyme for differential inhibition are considered. These mainly refer to the evidence that, although AR is an unspecific enzyme that recognizes different substrates such as aldoses and hydrophobic aldehydes, it nevertheless displays a certain degree of specificity among substrates of the same class. After screening on edible vegetables, indications of the presence of molecules potentially acting as ARDIs are reported.

The attempt to evaluate the human carbonyl reductase 1 (CBR1) activity on 3-glutathionylated-4-hydroxyalkanals through the classical spectrophotometric assay, in which NADPH oxidation is monitored at 340 nm, failed. This was due to the ability of the enzyme to catalyze the reduction of the free aldehyde form and at the same time the oxidation of the hemiacetal structure of this class of substrates, thus leading to the occurrence of a disproportion reaction sustained by a redox recycle of the pyridine cofactor. Making use of glutathionylated alkanals devoid of the 4 hydroxyl group, and thus unable to structurally arrange into a cyclic hemiacetal form, the susceptibility to inhibition of CBR1 to polyphenols was tested. Flavones, that were much more effective than isoflavones, resulted able to modulate the reductase activity of the enzyme on this new peculiar class of substrates.

Aldose reductase (AR) is an enzyme devoted to cell detoxification and at the same time is strongly involved in the aetiology of secondary diabetic complications and the amplification of inflammatory phenomena. AR is subjected to intense inhibition studies and dimethyl sulfoxide (DMSO) is often present in the assay mixture to keep the inhibitors in solution. DMSO was revealed to act as a weak but well detectable AR differential inhibitor, acting as a competitive inhibitor of the L-idose reduction, as a mixed type of non-competitive inhibitor of HNE reduction and being inactive towards 3-glutathionyl-4-hydroxynonanal transformation. A kinetic model of DMSO action with respect to differently acting inhibitors was analysed. Three AR inhibitors, namely the flavonoids neohesperidin dihydrochalcone, rutin and phloretin, were used to evaluate the effects of DMSO on the inhibition studies on the reduction of L-idose and HNE.

Carbonyl reductase 1 (CBR1) is an NADP-dependent enzyme that exerts a detoxifying role, which catalyses the transformation of carbonyl-containing compounds. The ability of CBR1 to act on adducts between glutathione and lipid peroxidation derived aldehydes has recently been reported. In the present study, exploiting mass spectrometry and fluorescence spectroscopy, evidence is shown that CBR1 is able to retain NADP(H) at the active site even after extensive dialysis, and that this retention may also occur when the enzyme is performing catalysis. This property, together with the multi-substrate specificity of CBR1 in both directions of red/ox reactions, generates inter-conversion red/ox cycles. This particular feature of CBR1, in the case of the transformation of 3-glutathionyl, 4-hydroxynonanal (GSHNE), which is a key substrate of the enzyme in detoxification, supports the disproportionation reaction of GSHNE without any apparent exchange of the cofactor with the solution. The importance of the cofactor as a prosthetic group for other dehydrogenases exerting a detoxification role is discussed.

The formation of the adduct between the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and glutathione, which leads to the generation of 3-glutathionyl-4-hydroxynonane (GSHNE), is one of the main routes of HNE detoxification. The aldo-keto reductase AKR1B1 is involved in the reduction of the aldehydic group of both HNE and GSHNE. In the present study, the effect of chirality on the recognition by aldose reductase of HNE and GSHNE was evaluated. AKR1B1 discriminates very modestly between the two possible enantiomers of HNE as substrates. Conversely, a combined kinetic analysis of the glutathionyl adducts obtained starting from either 4R- or 4S-HNE and mass spectrometry analysis of GSHNE products obtained from racemic HNE revealed that AKR1B1 possesses a marked preference toward the 3S,4R-GSHNE diastereoisomer. Density functional theory and molecular modeling studies revealed that this diastereoisomer, besides having a higher tendency to be in an open aldehydic form (the one recognized by AKR1B1) in solution than other GSHNE diastereoisomers, is further stabilized in its open form by a specific interaction with the enzyme active site. The relevance of this stereospecificity to the final metabolic fate of GSHNE is discussed.

The recruitment of the furanosidic scaffold of ribose as the crucial step for nucleotides and then for nucleic acids synthesis is presented. Based on the view that the selection of molecules to be used for relevant metabolic purposes must favor structurally well-defined molecules, the inadequacy of ribose as a preferential precursor for nucleotides synthesis is discussed. The low reliability of ribose in its furanosidic hemiacetal form must have played ab initio against the choice of d-ribose for the generation of d-ribose-5-phosphate, the fundamental precursor of the ribose moiety of nucleotides. The latter, which is instead generated through the ‘pentose phosphate pathway’ is strictly linked to the affordable and reliable pyranosidic structure of d-glucose.

Diabetes mellitus (DM) is a complex disease which currently affects more than 460 million people and is one of the leading cause of death worldwide. Its development implies numerous metabolic dysfunctions and the onset of hyperglycaemia-induced chronic complications. Multiple ligands can be rationally designed for the treatment of multifactorial diseases, such as DM, with the precise aim of simultaneously controlling multiple pathogenic mechanisms related to the disease and providing a more effective and safer therapeutic treatment compared to combinations of selective drugs. Starting from our previous findings that highlighted the possibility to target both aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two enzymes strictly implicated in the development of DM and its complications, we synthesised 3-(5-arylidene-4-oxothiazolidin-3-yl)propanoic acids and analogous 2-butenoic acid derivatives, with the aim of balancing the effectiveness of dual AR/PTP1B inhibitors which we had identified as designed multiple ligands (DMLs). Out of the tested compounds, 4f exhibited well-balanced AR/PTP1B inhibitory effects at low micromolar concentrations, along with interesting insulin-sensitizing activity in murine C2C12 cell cultures. The SARs here highlighted along with their rationalization by in silico docking experiments into both target enzymes provide further insights into this class of inhibitors for their development as potential DML antidiabetic candidates.

Background : The Zolfino bean is a variety of Phaseolus vulgaris , which is cultivated in a limited area of Tuscany, Italy, and is widely appreciated for its flavor and culinary uses. Objectives : A yellow Zolfino landrace cultivated in the Leccio-Reggello area was characterized and compared with three other varieties of Phaseolus vulgaris (i.e. the Borlotto, Cannellino, and Corona beans) in terms of its general features and potential as an antioxidant/anti-inflammatory agent. Design : The length, width, thickness, equatorial section surface, weight, volume, and seed coat section were measured in all the beans. The seed surface area was also estimated by an original empirical method. The ability of the different beans to interfere with the enzymes of the polyol pathway (that is, aldose reductase (AR) and sorbitol dehydrogenase) was tested using the supernatant after soaking the beans at room temperature and after thermal treatment, which simulated the bean-cooking process in a controlled fashion. Results : Concerning the general features, Zolfino was comparable with other beans, except Corona, in terms of surface–volume ratio, which possesses the lowest tegument thickness. Moreover, Zolfino appears the most effective in inhibiting AR activity. The inhibitory ability is unaffected by thermal treatment and appears to be associated with compound(s) present in the coat of the bean. Conclusions : The ability of Zolfino to inhibit AR, thus reducing the flux of glucose through the polyol pathway, highlights the features of Zolfino as a functional food, potentially useful in treating the dysfunctions linked to the hyperactivity of AR, such as diabetic complications or inflammatory responses. Keywords: Zolfino bean; Phaseolus vulgaris; aldose reductase; sorbitol dehydrogenase; polyol pathway (Published: 12 July 2016) Citation: Food & Nutrition Research 2016, 60: 31792 - http://dx.doi.org/10.3402/fnr.v60.31792

It is well known that a negative cooperative behavior displayed by a monomeric enzyme may be associated with the simultaneous presence of two enzymes acting on the same substrate. In this paper, emphasis is given to the effect exerted by a rapid equilibrium between the enzyme forms in leading to a hyperbolic behavior, thus masking the presence of multiple enzyme forms.

The chemical profile and the nutraceutical features of cultivated Salsola soda buds, in comparison with the wild plant, were investigated. Four flavonoids, rutin (1), quercetin 3-O-glucuronopyranoside (2), isorhamnetin 3-O-rutinoside (3), and isorhamnetin 3-O-glucuronopyranoside (4), were isolated from the wild S. soda and tested to target three human recombinant enzymes, i.e., aldose reductase (hAKR1B1), aldose-reductase-like protein (hAKR1B10), and carbonyl reductase 1 (hCBR1). Furthermore, three saponins, namely momordin IId (5), momordin IIc (6), and dihexosyl-pentosyl-glucuronosyl oleanolic acid (7) were identified in the plant and tested together for inhibitory activity of hAKR1B1. While no inhibitory activity was measured for the saponin mixture, flavonoids were shown to be able to inhibit the three target enzymes. Compound 2, the only flavonoid significantly represented in the cultivated edible S. soda, was shown to be the most effective inhibitor of the target enzymes. Indeed, it resulted in an appreciable inhibition of both hAKR1B1 and hCBR1, acting as an uncompetitive ( K i ' , 0.97 ± 0.24 μ M ) and as a mixed noncompetitive inhibitor (Ki, 0.84 ± 0.04; Ki’, 0.51 ± 0.05 μM), respectively. These results suggested the potential of S. soda components to favorably intervene in pathological conditions linked to diabetic complications, inflammatory processes, and in cancer therapy.

One of the consequences of the increased level of oxidative stress that often characterizes the cancer cell environment is the abnormal generation of lipid peroxidation products, above all 4-hydroxynonenal. The contribution of this aldehyde to the pathogenesis of several diseases is well known. In this study, we characterized the ADF astrocytoma cell line both in terms of its pattern of enzymatic activities devoted to 4-hydroxynonenal removal and its resistance to oxidative stress induced by exposure to hydrogen peroxide. A comparison with lens cell lines, which, due to the ocular function, are normally exposed to oxidative conditions is reported. Our results show that, overall, ADF cells counteract oxidative stress conditions better than normal cells, thus confirming the redox adaptation demonstrated for several cancer cells. In addition, the markedly high level of NADP+-dependent dehydrogenase activity acting on the glutahionyl-hydroxynonanal adduct detected in ADF cells may promote, at the same time, the detoxification and recovery of cell-reducing power in these cells.

While the evolutionary adaptation of enzymes to their own substrates is a well assessed and rationalized field, how molecules have been originally selected in order to initiate and assemble convenient metabolic pathways is a fascinating, but still debated argument. Aim of the present study is to give a rationale for the preferential selection of specific molecules to generate metabolic pathways. The comparison of structural features of molecules, through an inductive methodological approach, offer a reading key to cautiously propose a determining factor for their metabolic recruitment. Starting with some commonplaces occurring in the structural representation of relevant carbohydrates, such as glucose, fructose and ribose, arguments are presented in associating stable structural determinants of these molecules and their peculiar occurrence in metabolic pathways. Among other possible factors, the reliability of the structural asset of a molecule may be relevant or its selection among structurally and, a priori, functionally similar molecules.

4-hydroxynonenal (HNE) is one of the most abundant lipid peroxidation products and it is involved in the development of several pathological conditions. As other unsaturated lipid aldehydes, HNE readily reacts with glutathione, thus giving rise to the formation of 3-glutathionyl-4-hydroxynonanal (GSHNE). This step makes the removal of GSHNE, and in general of the glutathione-aldehydes adducts, a relevant pathway in lipid aldehydes detoxification. Human carbonyl reductase 1 (hCBR1), a member of the short chain dehydrogenase family, catalyses the reduction of glutathionylated aldehydes with an efficiency comparable to that observed for classical hCBR1 substrates. In the case of GSHNE, two functional groups are recognized by the catalytic action of hCBR1. In fact, the enzyme is able to both reduce the aldehydic group generating 3-glutathionyl-dihydroxynonane (GSDHN) and oxidize the hemiacetalic form generating 3-glutathionylnonanoic-γ-lactone. This disproportion reaction is supported by the recycle of the cofactor NADP+. Thus, hCBR1 may play a role in HNE detoxification, at the same time contributing to the recovery of reducing power. Moreover, the possibility to generate GSDHN, a signaling molecule that activates NFkB, makes hCBR1 possibly involved in the inflammation response.

Aldose reductase, the key enzyme of the polyol pathway, plays a crucial role in the development of long term complications affecting diabetic patients. Nevertheless, the expedience of inhibiting this enzyme to treat diabetic complications has failed, due to the emergence of side effects from compounds under development. Actually, aldose reductase is a Janus-faced enzyme which, besides ruling the polyol pathway, takes part to the antioxidant defence mechanism of the body. In this work we report the evidence that a novel class of compounds, characterized by a pyrazolo[1,5-a]pyrimidine core and a ionisable fragment, is able to modulate differently the catalytic activity of the enzyme, depending on the presence of specific substrates like sugar, toxic aldehydes and glutathione conjugates of toxic aldehydes. These compounds stand out as the first example of aldose reductase differential inhibitors (ARDIs), intended to target long term diabetic complications while leaving unaltered the detoxifying role of the enzyme.

Aldose reductase (ALR2) has been the target of therapeutic intervention for over 40 years; first, for its role in long-term diabetic complications and more recently as a key mediator in inflammation and cancer. However, efforts to prepare small-molecule aldose reductase inhibitors (ARIs) have mostly yielded carboxylic acids with rather poor pharmacokinetics. To address this limitation, the 1-hydroxypyrazole moiety has been previously established as a bioisostere of acetic acid in a group of aroyl-substituted pyrrolyl derivatives. In the present work, optimization of this new class of ARIs was achieved by the addition of a trifluoroacetyl group on the pyrrole ring. Eight novel compounds were synthesized and tested for their inhibitory activity towards ALR2 and selectivity against aldehyde reductase (ALR1). All compounds proved potent and selective inhibitors of ALR2 (IC50/ALR2 = 0.043−0.242 μΜ, Selectivity index = 190−858), whilst retaining a favorable physicochemical profile. The most active (4g) and selective (4d) compounds were further evaluated for their ability to inhibit sorbitol formation in rat lenses ex vivo and to exhibit substrate-specific inhibition.

Bovine lens aldose reductase is susceptible to a copper-mediated oxidation, leading to the generation of a disulfide bridge with the concomitant incorporation of two equivalents of the metal and inactivation of the enzyme. The metal complexed by the protein remains redox active, being able to catalyse the oxidation of different physiological thiol compounds. The thiol oxidase activity displayed by the enzymatic form carrying one equivalent of copper ion (Cu1-AR) has been characterized. The efficacy of Cu1-AR in catalysing thiol oxidation is essentially comparable to the free copper in terms of both thiol concentration and pH effect. On the contrary, the two catalysts are differently affected by temperature. The specificity of the AR-bound copper towards thiols is highlighted with Cu1-AR being completely ineffective in promoting the oxidation of both low-density lipoprotein and ascorbic acid.

Human recombinant carbonyl reductase 1 (E.C. 1.1.1.184, hCBR1) is shown to efficiently act as aldehyde reductase on glutathionylated alkanals, namely 3-glutathionyl-4-hydroxynonanal (GSHNE), 3-glutathionyl-nonanal, 3-glutathionyl-hexanal and 3-glutathionyl-propanal. The presence of the glutathionyl moiety appears as a necessary requirement for the susceptibility of these compounds to the NADPH-dependent reduction by hCBR1. In fact the corresponding alkanals and alkenals, and the cysteinyl and γ-glutamyl-cysteinyl alkanals adducts were either ineffective or very poorly active as CBR1 substrates. Mass spectrometry analysis reveals the ability of hCBR1 to reduce GSHNE to the corresponding GS-dihydroxynonane (GSDHN) and at the same time to catalyze the oxidation of the hemiacetal form of GSHNE, generating the 3-glutathionylnonanoic-δ-lactone. These data are indicative of the ability of the enzyme to catalyze a disproportion reaction of the substrate through the redox recycle of the pyridine cofactor. A rationale for the observed preferential activity of hCBR1 on different GSHNE diastereoisomers is given by molecular modelling. These results evidence the potential of hCBR1 acting on GSHNE to accomplish a dual role, both in terms of HNE detoxification and, through the production of GSDHN, in terms of involvement into the signalling cascade of the cellular inflammatory response.

A magnesium-dependent cysteinyl-glycine hydrolyzing enzyme from the gastropod mollusk Patella caerulea was purified to electrophoretic homogeneity through a simple and rapid purification protocol. The molecular masses of the native protein and the subunit suggest that the enzyme has a homohexameric structure. Structural data in combination with kinetic parameters determined with Cys-Gly and compared with Leu-Gly as a substrate, indicate that the purified enzyme is a member of the peptidase family M17. The finding that an enzyme of the peptidase family M17 is responsible also in mollusks for the breakdown of Cys-Gly confirms the important role of this peptidase family in the glutathione metabolism.

A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG.

Aldose reductase (AKR1B1), the key enzyme of the polyol pathway, plays a crucial role in the development of long-term complications affecting diabetic patients. Nevertheless, the expedience of inhibiting this enzyme to treat diabetic complications has failed, due to the emergence of side effects from compounds under development. Actually AKR1B1 is a Janus-faced enzyme which, besides ruling the polyol pathway, takes part in the antioxidant defense mechanism of the body. In this work we report the evidence that a class of compounds, characterized by a pyrazolo[1,5-a]pyrimidine core and an ionizable fragment, modulates differently the catalytic activity of the enzyme, depending on the presence of specific substrates such as sugar, toxic aldehydes, and glutathione conjugates of toxic aldehydes. The study stands out as a systematic attempt to generate aldose reductase differential inhibitors (ARDIs) intended to target long-term diabetic complications while leaving unaltered the detoxifying role of the enzyme.

4-Hydroxy-2-nonenal (HNE) is one of the main products of lipid autoxidation of unsaturated fatty acid (1). It is a highly reactive molecule and has received a particular attention for its biological activity and its role in different diseases (2). HNE metabolism is reported to mainly occur through its conjugation with glutathione and the subsequent formation of 3-glutathionyl-4-hydroxynonanal (GSHNE) (3). This molecule is susceptible to both reductive and oxidative transformations, which occur through the action of either the NADPH-dependent action of aldose reductase or the NADP+-dependent activity of aldehyde dehydrogenase, respectively. In this context it has been identified a role of carbonyl reductase 1 (CBR1) in the detoxification of GSHNE through its oxidation to the corresponding 3-glutathionyl-nonanoic-δ-lactone (4). More recently it has been also reported the capability of the enzyme to reduce GSHNE, together with a number of glutathionylated aldehydes, to the corresponding GS-dihydroxynonane (GSDHN) (5). Thus GSHNE, through its equilibrium between the open aldehyde form and its cyclic hemiacetal acts, with respect to CBR1, as a dual substrate. Being the two red/ox processes linked to the same red/ox cofactor (NADP+/NADPH), it turns out that a CBR1-catalyzed disproportion of GSHNE occurs. The high catalytic efficiency of CBR1 in the GSHNE processing, would suggest this molecule as one of the main physiological substrate of the enzyme. In this contest besides contributing to detoxification processes, CBR1 may be involved in the production of a signaling molecule, GSDHN, which is reported to activate inflammatory processes mediated by NFκB (6). These results could add new relevance on the inhibition of CBR1 activity by specific molecules that could play an anti-inflammatory role.

An NADP(+)-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM 33 µM, kcat 405 min(-1)), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic-δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.

Background Glucose is considered as one of the main sources of cell damage related to aldose reductase (AR) action in hyperglycemic conditions and a worldwide effort is posed in searching for specific inhibitors of the enzyme. This AR substrate has often been reported as generating non-hyperbolic kinetics, mimicking a negative cooperative behavior. This feature was explained by the simultaneous action of two enzyme forms acting on the same substrate. Methods The reduction of different aldoses and other classical AR substrates was studied using pure preparations of bovine lens and human recombinant AR. Results The apparent cooperative behavior of AR acting on glucose and other hexoses and pentoses, but not on tethroses, glyceraldehyde, 4-hydroxynonenal and 4-nitrobenzaldehyde, is generated by a partial nonclassical competitive inhibition exerted by the aldose hemiacetal on the reduction of the free aldehyde. A kinetic model is proposed and kinetic parameters are determined for the reduction of l -idose. Conclusions Due to the unavoidable presence of the hemiacetal, glucose reduction by AR occurs under different conditions with respect to other relevant AR-substrates, such as alkanals and alkenals, coming from membrane lipid peroxidation. This may have implications in searching for AR inhibitors. The emerging kinetic parameters for the aldoses free aldehyde indicate the remarkable ability of the enzyme to interact and reduce highly hydrophilic and bulky substrates. General significance The discovery of aldose reductase modulation by hemiacetals offers a new perspective in searching for aldose reductase inhibitors to be developed as drugs counteracting the onset of diabetic complications.

The chaperone behaviour of bovine serum albumin was compared with that of alpha-crystallin. The chaperone activity was assessed by measuring: (i) the ability to antagonize protein aggregation induced by heat; (ii) the capability to protect the activity of thermally stressed enzymes and (iii) the effectiveness in assisting the functional recovery of chemically denatured sorbitol dehydrogenase. Despite the lack of structural analogies, both proteins show several functional similarities in preventing inactivation of thermally stressed enzymes and in reactivating chemically denatured sorbitol dehydrogenase. As with alpha-crystallin, the chaperone action of bovine serum albumin appears to be ATP independent. Bovine serum albumin appears significantly less effective than alpha-crystallin only in preventing thermally induced protein aggregation. A possible relationship between chaperone function and structural organization is proposed. Together, our results indicate that bovine serum albumin acts as a molecular chaperone and that, for its particular distribution, can be included in the extracellular chaperone family.

Although glucose is one of the most important physio-pathological substrates of aldose reductase, it is not an easy molecule for in vitro investigation into the enzyme. In many cases alternative aldoses have been used for kinetic characterization and inhibition studies. However these molecules do not completely match the structural features of glucose, thus possibly leading to results that are not fully applicable to glucose. We show how aldose reductase is able to act efficiently on L-idose, the C-5 epimer of D-glucose. This is verified using both the bovine lens and the human recombinant enzymes. While the kcat values obtained are essentially identical to those measured for D-glucose, a significant decrease in KM was observed. This can be due to the significantly higher level of the free aldehyde form present in L-idose compared to D-glucose. We believe that L-idose is the best alternative to D-glucose in studies on aldose reductase.

The possible preferential action exerted by an inhibitor on the transformation of one of two agonist substrates catalyzed by the same enzyme has recently been reported in studies on aldose reductase inhibition. This event was defined as “intra-site differential inhibition” and the molecules able to exert this action as “differential inhibitors”. This work presents some basic kinetic models describing differential inhibition. Using a simple analytic approach, the results show that differential inhibition can occur through either competitive or mixed type inhibition in which the inhibitor prevalently targets the free enzyme. The results may help in selecting molecules whose differential inhibitory action could be advantageous in controlling the activity of enzymes acting on more than one substrate.

Sorbitol dehydrogenase (l-iditol:NAD(+) 2-oxidoreductase, E.C. 1.1.1. 14) (SDH) was significantly protected from thermally induced inactivation and aggregation by bovine lens alpha-crystallin. An alpha-crystallin/SDH ratio as low as 1:2 in weight was sufficient to preserve the transparency of the enzyme solution kept for at least 2 h at 55 degrees C. Moreover, an alpha-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55 degrees C for at least 40 min. The protection by alpha-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5 m NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the alpha-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by alpha-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of alpha-crystallin against the inactivation of SDH induced at 55 degrees C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of alpha-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with alpha-crystallin. Two different adducts between alpha-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a high M(r) of approximately 800,000 and carried exclusively inactive SDH. A second adduct, carrying active SDH, had a size consistent with an interaction of the enzyme with monomers or low M(r) aggregates of alpha-crystallin. Even though it had a reduced efficiency with respect to alpha-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of alpha-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of alpha-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.

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.

2-Thioxo-4-thiazolidinone derivatives were evaluated as aldose reductase inhibitors (ARIs) and most of them exhibited good or excellent in vitro efficacy. Out of the tested compounds, most N-unsubstituted analogues were found to possess inhibitory effects at low micromolar doses and two of them exhibited higher potency than sorbinil, used as a reference drug. The insertion of an acetic chain on N-3 of the thiazolidinone scaffold led to analogues with submicromolar affinity for ALR2 and IC 50 values very similar to that of epalrestat, the only ARI currently used in therapy.

Non-carboxylic acid containing bioisosteres of (5-arylidene-2,4-dioxothiazolidin-3-yl)acetic acids, which are active as aldose reductase (ALR2) inhibitors, were designed by replacing the carboxylic group with the trifluoromethyl ketone moiety. The in vitro evaluation of the ALR2 inhibitory effects of these trifluoromethyl substituted derivatives led to the identification of two inhibitors effective at low micromolar doses. It was further confirmed that a carboxylic chain on N-3 of the thiazolidinedione scaffold is a determining requisite to obtain the highest efficacy levels; however, it is not essential for the interaction with the target enzyme and it can be replaced by different polar groups, thus obtaining less ionised or unionised inhibitors.

Purpose To define a possible metabolic and/or signaling role for Cys-Gly in glutathione homeostasis in bovine eye lenses. Methods Bovine lenses were cultured up to 24 h in a medium containing 0.5 mM reduced glutathione (GSH) under different conditions. The intracellular and the extracellular contents of thiol compounds were evaluated using a free zone capillary electrophoresis method. Results Culture of lenses in the presence of GSH and the gamma-glutamyl transferase inhibitor serine-borate demonstrated a 1.5 fold increase in the level of extra-lenticular glutathione with respect to the initial value. Cys-Gly exogenously added impaired the extra-lenticular accumulation of glutathione. Both cysteine and gamma-Glu-Cys were ineffective in reducing extra-lenticular glutathione accumulation. In all conditions no differences in reduced and total intra-lenticular glutathione levels were observed. Conclusions The impairment of Cys-Gly generation correlated with inhibition of gamma-glutamyl transferase by serine/borate, resulting in high extra-lenticular concentration of glutathione effluxed from the bovine lens. The possibility that Cys-Gly may intervene either in the replenishment processes for cysteine in the GSH biosynthetic step or in the function of the efflux GSH-transporters is considered.

A colorimetric coupled enzyme assay for the determination of gamma-glutamyltransferase (GGT) activity using glutathione as substrate is described. The cysteine released from glutathione upon sequential action of GGT and leucine aminopeptidase is spectrophotometrically detected through its reaction with ninhydrin at 100 degrees C in acidic conditions. The method was applied to the determination of the activity of both bovine kidney and human serum GGT. In the described assay conditions with final GGT concentrations ranging from 0.18 to 4 mU/ml, a linear relationship between produced cysteine and incubation times up to 90 min was observed. When a standard chromogenic assay for GGT using L-gamma-glutamyl-3-carboxy-4-nitroanilide as substrate and the proposed assay were applied on the same serum sample a linear relationship between the two method was observed. Since the use of GSH as substrate, the proposed method can be usefully adopted for enzymological studies on GGT-related enzymes, a class of enzymes which is still waiting to be characterized.

The ability of Zofenoprilat, an angiotensin-converting enzyme inhibitor carrying a thiol group, to intervene in protein S-thiolation processes was tested on bovine lens aldose reductase (ALR2). Zofenoprilat, more susceptible to oxidation than glutathione (GSH), forms with this physiological thiol a rather stable mixed disulfide (ZSSG). ZSSG, whose generation through the transthiolation reaction between GSH and Zofenoprilat homodisulfide was shown to be enhanced by a micro-class glutathione S-transferase, appears to be a specific donor of the Zofenoprilat moiety in the S-thiolation processes. This is indicated by the apparent stability of ZSSG to reduction by GSH and by the specificity of the transfer of the group on ALR2, used as a protein model. Indeed, the S-thiolation of ALR2 by ZSSG occurred exclusively through the insertion of the Zofenoprilat moiety of ZSSG on the enzyme. The modified ALR2 is shown to retain the same activity of the native enzyme, but displays a reduced sensitivity to inhibition. The S-thiolation of specific target enzymes is proposed as an event potentially relevant for the antioxidant action of Zofenoprilat.