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A chaperone-like activity of bovine lens alpha-crystallin against the thermal-induced aggregation of bovine lens aldose reductase is reported. While the precipitation of aldose reductase at 55 degrees C is prevented by alpha-crystallin present at a ratio of aldose reductase: alpha-crystallin as low as 1:0.5 (w:w), the heat-induced inactivation of the enzyme occurs regardless of the presence of alpha-crystallin. This would suggest that, irrespective of the functional integrity of the target protein, alpha-crystallin interferes only with aggregation phenomena, having the potential to preserve the lens transparency. Calcium and magnesium ions at mM levels affect the antiaggregation action exerted by alpha-crystallin either interfering on the formation or reducing the stability of the aldose reductase: alpha-crystallin complex.

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.

Bovine lens aldose reductase (alditol: NADP+ oxidoreductase, EC 1.1.1.21) undergoes a modification induced by 2-mercaptoethanol in the presence of the redox system Fe(II)/Fe(III). The modified form (ARa) exhibits an increased hydrophobicity and tendency to aggregate. Moreover, while the native enzyme form is rather insensitive to proteolytic breakdown, the modified form is susceptible to limited proteolysis by trypsin and chymotrypsin. With both proteases, the degradation correlated with a loss of enzyme activity and results in the appearance of one molecular species of 26 KDa (for chymotrypsin) and two molecular species of 24 and 17 KDa (for trypsin). The decline in solubility and the increase in susceptibility to proteolysis of ARa suggests that the thiol-dependent metal-catalyzed modification is comparable to other oxidative systems that mark proteins for degradation.