Complete protection by alpha-crystallin of lens sorbitol dehydrogenase undergoing thermal stress

Sorbitol dehydrogenase (L-iditol:NAD(+) 2-oxidoreductase, E.C. 1.1.1.14) (S DH) was significantly protected from thermally induced inactivation and agg regation by bovine lens cu-crystallin, An alpha -crystallin/SDH ratio as lo w as 1:2 in weight was sufficient to preserve the transparency of the enzym e solution kept for at least 2 h at 55 degreesC, Moreover, an alpha -crysta llin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55 degreesC for at least 40 min. The protection by alpha -crystall in 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 wa s lost at a high salt concentration because of the precipitation of the alp ha -crystallin/SDH adduct, Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by alpha -crystal lin against the thermally induced inactivation and aggregation of SDH, The lack of protection of alpha -crystallin against the inactivation of SDH ind uced at 55 degreesC by thiol blocking agents or EDTA together with the addi tive effect of NADH in stabilizing the enzyme in the presence of rw-crystal lin suggest that functional groups involved in catalysis are freely accessi ble in SDH while interacting with alpha -crystallin. Two different adducts between alpha -crystallin and SDH were isolated by gel filtration chromatog raphy, 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 l ow M,aggregates of alpha -crystallin. Even though it had a reduced efficien cy with respect to alpha -crystallin, bovine serum albumin was shown to mim ic 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 t he stabilization of the enzyme activity.