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) (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.
Università di Pisa, Dipartimento di Fisiologia e Biochimica, Laboratorio di Biochimica, via S. Maria 55, 56100 Pisa, Italy.
I
I
Marini
R
R
Moschini
A
A
Del Corso
U
U
Mura
D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002417:Cattle; D002850:Chromatography, Gel; D003459:Crystallins / Q000737:chemistry* / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D007064:L-Iditol 2-Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007908:Lens, Crystalline / Q000201:enzymology*; D015636:Magnesium Chloride / Q000494:pharmacology; D012996:Solutions; D013816:Thermodynamics
D003459:Crystallins; D012996:Solutions; D015636:Magnesium Chloride; D007064:L-Iditol 2-Dehydrogenase; D002122:Calcium Chloride