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.Aldose reductase (alditol:NADP oxidoreductase, EC 1.1.1.21) (ALR2) 1 catalyzes with a broad catalytic efficiency the NADPHdependent reduction of aldo-sugars and a variety of aromatic and aliphatic aldehydes to their corresponding alcohols. This enzyme is the first in a pathway that results in the transformation of glucose to fructose using sorbitol as a metabolic intermediate. This so-called "polyol pathway" is not a "high flux" metabolic route except in hyperglycemic conditions such as diabetes mellitus and galactosemia, where elevated concentrations of glucose and galactose, respectively, result in enhanced accumulation of their corresponding polyols in various tissues such as the eye lens (1, 2). Since these polyols do not readily permeate cell membranes, their intracellular accumulation is thought to create an osmotic imbalance, resulting ultimately in sugar cataract formation (3-5). Intensive effort has been mounted to identify inhibitors of aldose reductase for use as therapeutic tools against diabetic complications such as cataract and retinopathy (6 -9).Aldose reductase is subject to modifications leading to enzyme forms with an altered sensitivity to various inhibitors. Thus, the so-called "activated" ALR2 generated through apparently different processes such as isomerization (10), glycosylation (11), and thiol-dependent oxidation (12-14), besides displaying differences in substrate specificity, has a greatly reduced sensitivity to different aldose reductase inhibitors. Indeed, others recently reported the purification of human ALR2 with kinetic properties consistent with those described for an oxidized form of the enzyme (15). The potential involvement of cysteine residues in catalysis ...

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.

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 ␣-crystallin. An ␣-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°C. Moreover, an ␣-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55°C for at least 40 min. The protection by ␣-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 ␣-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by ␣-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of ␣-crystallin against the inactivation of SDH induced at 55°C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of ␣-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with ␣-crystallin. Two different adducts between ␣-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 ␣-crystallin. Even though it had a reduced efficiency with respect to ␣-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of ␣-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of ␣-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.

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