THE 2 FORMS OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE ACTIVASE DIFFER IN SENSITIVITY TO ELEVATED-TEMPERATURE/

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase often consist s of two polypeptides that arise from alternative splicing of pre-mRNA . In this study recombinant versions of the spinach (Spinacea oleracea L.) 45- and 41-kD forms of activase were analyzed for their response to temperature. The temperature optimum for ATP hydrolysis by the 45-k D form was 45 degrees C, approximately 13 degrees C higher than the 41 -kD form. When the two forms were mixed, the temperature response of t he hybrid enzyme was similar to the 45-kD form. In the absence of aden ine nucleotide, preincubation of either activase form at temperatures above 25 degrees C inactivated ATPase activity. Adenosine 5'-(gamma-th io)triphosphate, but not ADP, significantly enhanced the thermostabili ty of the 45-kD form but was much less effective for the 41-kD form. I ntrinsic fluorescence showed that the adenosine 5'-(gamma-thio)triphos phate-induced subunit aggregation was lost at a much lower temperature for the 41-kD than for the 45-kD form. However, the two activase form s were equally susceptible to limited proteolysis after heat treatment . The results indicate that (a) the 45-kD form is more thermostable th an, and confers increased thermal stability to, the 41-kD form, and (b ) a loss of subunit interactions, rather than enzyme denaturation, app ears to be the initial cause of temperature inactivation of activase.