The Two Forms of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase Differ in Sensitivity to Elevated Temperature

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase often consists 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-kD form was 45[deg]C, approximately 13[deg]C higher than the 41-kD form. When the two forms were mixed, the temperature response of the hybrid enzyme was similar to the 45-kD form. In the absence of adenine nucleotide, preincubation of either activase form at temperatures above 25[deg}C inactivated ATPase activity. Adenosine 5[prime]-([gamma]-thio)triphosphate, but not ADP, significantly enhanced the thermostability of the 45-kD form but was much less effective for the 41-kD form. Intrinsic fluorescence showed that the adenosine 5[prime]-([gamma]-thio)triphosphate-induced subunit aggregation was lost at a much lower temperature for the 41-kD than for the 45-kD form. However, the two activase forms were equally susceptible to limited proteolysis after heat treatment. The results indicate that (a) the 45-kD form is more thermostable than, and confers increased thermal stability to, the 41-kD form, and (b) a loss of subunit interactions, rather than enzyme denaturation, appears to be the initial cause of temperature inactivation of activase.