THERMAL INACTIVATION AND CHAPERONIN-MEDIATED RENATURATION OF MITOCHONDRIAL ASPARTATE-AMINOTRANSFERASE

Mitochondrial aspartate aminotransferase is inactivated irreversibly o n heating. The inactivated protein aggregates, but aggregation is prev ented by the presence of the chaperonin 60 from Escherichia coli (GroE L). The chaperonin increases the rate of thermal inactivation in the t emperature range 55-65 degrees C but not at lower temperatures. It has previously been shown [Twomey and Doonan (1997) Biochim. Biophys. Act a 1342, 37-44] that the enzyme switches to a modified, but catalytical ly active, conformation at approx. 55-60 degrees C and the present res ults show that this conformation is recognized by and binds to GroEL. The thermally inactivated protein can be released from GroEL in an act ive form by the addition of chaperonin 10 from E. coli (GroES)/ATP, sh owing that inactivation is not the result of irreversible chemical cha nges. These results suggest that the irreversibility of thermal inacti vation is due to the formation of an altered conformation with a high kinetic barrier to refolding rather than to any covalent changes. In t he absence of chaperonin the unfolded molecules aggregate but this is a consequence, rather than the cause, of irreversible inactivation.