Signalling potential and protein modifying ability of physiological thiols.
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Aldehyde Reductase↗
Biomedical subjects
M Cappiello
Publications and source records attributed to M Cappiello.
Thiol/disulfide interconversion in bovine lens aldose reductase induced by intermediates of glutathione turnover.
The effectiveness of cysteine and cysteinylglycine to act as protein thiolating agents was investigated using bovine lens aldose reductase (ALR2) as the protein target. Disulfides of both thiol compounds appear to be very effective as ALR2 thiolating agents. Cysteine- and CysGly-modified ALR2 forms (Cys-ALR2 and CysGly-ALR2, respectively) are characterized by the presence of a mixed disulfide bond involving Cys298, as demonstrated by a combined electrospray mass spectrometry and Edman degradation approach. Both Cys-ALR2 and CysGly-ALR2 essentially retain the ability to reduce glyceraldehyde but lose the susceptibility to inhibition by Sorbinil and other ALR2 inhibitors. Cys-ALR2 and CysGly-ALR2 are easily reduced back to the native enzyme form by dithiothreitol and GSH treatment; on the contrary, Cys and 2-mercaptoethanol appear to act as protein trans-thiolating agents, rather than reducing agents. The treatment at 37 degrees C of both Cys-ALR2 and CysGly-ALR2, unlikely what observed for glutathionyl-modified ALR2 (GS-ALR2), promotes the generation of an intramolecular disulfide bond between Cys298 and Cys303 residues. A rationale for the special susceptibility of Cys-ALR2 and CysGly-ALR2, as compared to GS-ALR2, to the thermally induced intramolecular rearrangement is given on the basis of a molecular dynamic and energy minimization approach. A pathway of thiol/disulfide interconversion for bovine lens ALR2 induced, in oxidative conditions, by physiological thiol compounds is proposed.
Aldehyde Reductase↗
Modulation of aldose reductase activity through S-thiolation by physiological thiols.
The glutathionyl-modified aldose reductase (GS-ALR2) is unique, among different S-thiolated enzyme forms, in that it displays a lower specific activity than the native enzyme (ALR2). Specific interactions of the bound glutathionyl moiety (GS) with the ALR2 active site, were predicted by a low perturbative molecular modelling approach. The outcoming GS allocation, involving interactions with residues relevant for catalysis and substrate allocation, explains the rationale behind the observed differences in the activity between GS-ALR2 and other thiol-modified enzyme forms. The reversible S-glutathionylation of ALR2 observed in cultured intact bovine lens undergoing an oxidative/non oxidative treatment cycle is discussed in terms of the potential of ALR2/GS-ALR2 inter-conversion as a response to oxidative stress conditions.
Aldehyde Reductase↗
NAAG inhibits KCl-induced [(3)H]-GABA release via mGluR3, cAMP, PKA and L-type calcium conductance.
The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective agonist at the type 3 metabotropic glutamate receptor (mGluR3) where it acts to decrease cAMP levels. Rat cortical interneurons express both NAAG and glutamic acid decarboxylase, as well as mGluR3 mRNA. In the presence of ionotropic glutamate receptor antagonists, both NAAG and the group II metabotropic glutamate receptor agonist, DCG-IV, reduced the calcium-dependent, KCl-induced [(3)H]-GABA release from rat cortical neurons by 35%. This release process was unaffected by tetrodotoxin. The group II antagonist, ethyl glutamate, reversed the effects of DCG-IV and NAAG. The mGluR3-selective antagonist, beta-N-acetylaspartylglutamate, reversed the effect of NAAG. While pretreatment of cortical neurons with forskolin alone did not significantly affect KCl-stimulated [(3)H]-GABA-release, forskolin abolished the inhibition of release produced by NAAG. The protein kinase A inhibitor, H-89, decreased [(3)H]-GABA release while NAAG produced no additional inhibition in the presence of H-89. In contrast, the protein kinase C inhibitor, Ro 31--8220, had no effect on KCl-stimulated release, nor did it affect the inhibition of release produced by NAAG. The L-type calcium channel blocker, nifedipine, also inhibited the release of [(3)H]-GABA and coapplication with NAAG resulted in no significant additional inhibition of release. These data support the hypothesis that the inhibition of KCl-stimulated [(3)H]-GABA release by NAAG is mediated via presynaptic mGluR3 on GABAergic cortical neurons and that this effect is obtained by decreasing cAMP with a consequent decrease in protein kinase A activity and L-type calcium channel conductance.
Animals↗
In vitro recycling of alpha-D-ribose 1-phosphate for the salvage of purine bases.
In this paper, we extend our previous observation on the mobilization of the ribose moiety from a purine nucleoside to a pyrimidine base, with subsequent pyrimidine nucleotides formation (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273-281). The data show that, at least in vitro, also the reverse process is possible. In rat brain extracts, the activated ribose, stemming from uridine as ribose 1-phosphate, can be used to salvage adenine and hypoxanthine to their respective nucleotides. Since the salvage of purine bases is a 5-phosphoribosyl 1-pyrophosphate-dependent process, catalyzed by adenine phosphoribosyltransferase and hypoxanthine guanine phosphoribosyltransferase, our results imply that Rib-1P must be transformed into 5-phosphoribosyl 1-pyrophosphate, via the successive action of phosphopentomutase and 5-phosphoribosyl 1-pyrophosphate synthetase; and,in fact, no adenosine could be found as an intermediate when rat brain extracts were incubated with adenine, Rib-1P and ATP, showing that adenine salvage does not imply adenine ribosylation, followed by adenosine phosphorylation. Taken together with our previous results on the Rib-1P-dependent salvage of pyrimidine nucleotides, our results give a clear picture of the in vitro Rib-1P recycling, for both purine and pyrimidine salvage.
Animals↗
Thiol disulfide exchange modulates the activity of aldose reductase in intact bovine lens as a response to oxidative stress.
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.
Aldehyde Reductase↗
Ribose 1-phosphate and inosine activate uracil salvage in rat brain.
The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.
Animals↗
A new approach against sugar cataract through aldose reductase inhibitors.
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.
Aldehyde Reductase↗
Interconversion pathways of aldose reductase induced by thiol compounds.
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Aldehyde Reductase↗
In vitro assessment of salvage pathways for pyrimidine bases in rat liver and brain.
In this paper we extend our previous observation on the mobilization of the ribose moiety from guanosine to xanthine catalyzed by rat liver extracts (Giorgelli et al., Biochim. Biophys. Acta 1335 (1997) 16-22). The data show that in rat liver and brain extracts the activated ribose, stemming from inosine and guanosine phosphorolysis as ribose 1-phosphate, can be used to salvage uracil to uracil nucleotides. Uridine is an intermediate. The salvage process occurs even in the presence of excess inorganic phosphate suggesting that uridine phosphorylase may function in vivo as an anabolic enzyme. Ribose 5-phosphate cannot substitute for inosine, guanosine or ribose 1-phosphate as ribose donor. When inorganic phosphate was substituted with arsenate, hindering the formation of ribose 1-phosphate, no ribose transfer could be observed. A similar pathway occurs at the deoxy level. The deoxyribose moiety of deoxyinosine can be used to salvage thymine to thymine nucleotides, again in the presence of excess inorganic phosphate. Our results introduce a novel aspect of the salvage pathway, in which ribose 1-phosphate seems to play a pivotal role.
Animals↗
Site-specific inactivation of aldose reductase by 4-hydroxynonenal.
Bovine lens aldose reductase (ALR2), which catalyzes the NADPH-dependent reduction of 4-hydroxy-2-nonenal (HNE), is readily inactivated by its own substrate in a time- and concentration-dependent manner. Both DTT and NADP+ can prevent enzyme inactivation but neither extensive dialysis nor thiol-reducing treatment were able to restore enzyme activity once inactivation had occurred. Unlike the native enzyme, S-glutathionyl-modified ALR2 is unaffected by HNE, and can be easily reverted to the native form under thiol-reducing conditions. Evidence is presented of the involvement of Cys298 in the inactivation process. Zofenoprilat, an antioxidant thiol compound, mimics the effect of GSH. The possibility is raised that enzyme thiolation may function as a protection mechanism against the irreversible modification of ALR2.
Aldehyde Reductase↗
Hirunorms, novel hirudin-like direct thrombin inhibitors.
1. Hirunorms are new synthetic peptides designed to interact with thrombin in a similar way to the natural inhibitor hirudin. 2. Hirunorms are specific and efficient in vitro inhibitors of thrombin activity. 3. Hirunorms are potent anticoagulant and antithrombotic agents in in vivo experimental models devoid of hemorrhagic effects at doses that are active in preventing thrombosis.
Antithrombins↗
Predictors of attendance at a relocatable mammography service for rural women.
This study aimed to identify factors that predicted attendance at a relocatable screening mammography service in a rural centre in Victoria. A cohort design was used whereby 180 women from the target population were interviewed by telephone two weeks before the service moved to the area for a 10-week period of operation. Attendance data were ascertained from service records. Fifty per cent of the sample attended the service. Significant predictors of attendance were: mammographic history, with women who reported previous screening mammography being less likely to attend than women who had not had a previous mammogram (odds ratio (OR) 0.38, 95% confidence interval (CI) 0.17 to 0.83); perception of personal risk for breast cancer, with women who perceived at least some risk being more likely to attend than women who perceived no risk (OR 2.73, CI 1.07 to 6.99); stated intention of attending (OR 2.01, CI 1.49 to 2.71); knowing the correct location of the service (OR 3.08, CI 1.37 to 6.89); and education, with higher education being associated with a lower likelihood of attending (OR 0.65, CI 0.44 to 0.96). Our study raised some issues, including the high prevalence of rural women who reported a previous screening mammogram, although BreastScreen services had not previously been available in their area; factors underlying perceptions of personal risk for breast cancer; and the generalisability of our finding of an inverse relationship between higher education and attendance for screening.
Aged↗
Leaving work to smoke.
Work-place smoking bans have not only reduced work-day cigarette consumption but also been associated with going outside to smoke during working hours. We examined the extent of "exiled smoking", estimated how much work-day cigarette consumption can be attributed to it, and examined proximal predictors of both these two variables. Some 794 smokers from 42 medium-sized work-places were surveyed as the baseline for an intervention study. A self-completed questionnaire assessed smoking behaviour on work and non-working days, leaving work to smoke, and beliefs and opinions about smoking and smoking bans. Multiple regressions were used to examine predictors of leaving work to smoke, and of the amount smoked when doing so. Smokers reported consuming an average of 5.4 cigarettes during work breaks, 3.5 of which were associated with deliberately seeking opportunities to smoke; 39% reported leaving work to smoke one or more times per day during non-break periods. Indices of addiction were significant predictors of both leaving work to smoke and of cigarette consumption while doing so. Leaving work to smoke is in part an activity of addicted smokers, presumably to maintain blood nicotine levels. There is the potential to further reduce rates of cigarette consumption associated with work-place smoking bans if this "exiled smoking" can be reduced. This may be easier to achieve in light smokers.
Adult↗
Specifically targeted modification of human aldose reductase by physiological disulfides.
Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.
Aldehyde Reductase↗
Kinetics of human thrombin inhibition by two novel peptide inhibitors (Hirunorm IV and Hirunorm V).
A study on the kinetics of human thrombin inhibition by two novel synthetic peptides (Hirunorm IV and Hirunorm V) and a comparison with recombinant hirudin and a commonly used thrombin inhibitor, Hirulog-1, are reported. The dissociation constants for Hirunorm IV and Hirunorm V were determined by varying the concentration of inhibitors at fixed concentrations of the chromogenic substrate Chromozym-TH (N-tosylglycyl-L-prolyl-L-arginine 4-nitroanilide acetate). Both inhibitors behaved as reversible tight-binding inhibitors of amidolytic thrombin activity. The apparent dissociation constants determined showed a linear dependence on the concentration of substrate; this finding, which indicates that the inhibition was competitive, made possible the estimation of the dissociation constants (KI) for Hirunorm IV and Hirunorm V, which were 0.134 +/- 0.014 nM and 0.245 +/- 0.016 nM, respectively. Similar dissociation constants were also obtained for the two inhibitors when thrombin activity was measured with fibrinogen in the clotting assay. When tested for resistance to thrombin proteolytic activity, both inhibitors were inviolate to cleavage by thrombin. The data obtained demonstrate that both Hirunorm IV and Hirunorm V are potent and stable inhibitors of human thrombin activity.
Amino Acid Sequence↗
Synthesis, activity, and molecular modeling of a new series of tricyclic pyridazinones as selective aldose reductase inhibitors.
Three new series of tricyclic pyridazinones have been synthesized and tested in vitro in order to assess (i) their ability to inhibit aldose reductase enzyme (ALR2) and (ii) their specificity toward the target enzyme with respect to other related oxidoreductases, such as aldehyde reductase, sorbitol dehydrogenase, and glutathione reductase. The inhibitory capability of the most effective compounds (IC50 values ranging from 6.44 to 12.6 microM) appears to be associated with a rather significant specificity for ALR2. Molecular mechanics and molecular dynamic calculations performed on the ALR2-inhibitor complex give indications of specific interaction sites responsible for the binding, thus providing information for the design of new inhibitors with improved affinity for the enzyme.
Aldehyde Reductase↗
Occurrence of glutathione-modified aldose reductase in oxidatively stressed bovine lens.
The optimization of an affinity chromatography method on Matrex Orange resin allowed the separation of glutathione modified and native aldose reductase in crude extracts of bovine lens. The analysis of hyperbaric oxygen treated lenses revealed the formation in the intact cultured lens of an enzyme form displaying affinity column binding properties, specific activity, sensitivity to inhibition and susceptibility to activation by thiol reducing agents, all comparable to glutathione modified aldose reductase. The extent of the enzyme modification increased with the time of the oxidative treatment and was maximal in the lens nucleus. The relative increase of glutathione modified aldose reductase from cortex to the nucleus is consistent with the increase in these lens regions of the GSSG/GSH ratio.
Aerobiosis↗