Frozen Denatured EnzymesRefoldingAetivity

We found that a cold acclimation protein from an ice-nucleating bacterium, lhtoea ananas KUIN-3, has refolding actiyity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the co]d shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refo]ding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH4)2S04 precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,OOe each with a molecular mass of 200,OOO and had refolding activity against denatured enzymes, which were denat"red by heat-treatment at 1000C, cryopreservation at -20eC, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was MetArg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high leye) of activity at low temperature (12eC). A]so, the dissociation constants, K) (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82 × 10-iO, 4.35× 10m9, 8.98× 10-i2, and 3.05 × 10-ii, respectively. The aMnity of Hsc25 for frozen danatured enzymes was higher than the aMnity for heat denatured enzymes when compared with tke aMnity ef GreEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.