The Two Forms of Ribulose-I ,5=Bisphosphate Carboxylase/Oxygenase Activase Differ in Sensitivity to

Ribulose-l,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.) 45and 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”C, approximately 13°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°C inactivated ATPase activity. Adenosine 5’-(y-thio)triphosphate, but not ADP, significantly enhanced the thermostability of the 45-kD form but was much less effective for the 41-kD form. lntrinsic fluorescence showed that the adenosine 5’-(y-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. Zielinski, 1991a). In spinach the 45and 41-kD activase polypeptides are identical except for 37 additional amino acids on the C-terminal end of the 45-kD form. Both the 45and the 41-kD forms of spinach activase catalyze ATP hydrolysis, as well as activate Rubisco (Shen et al., 1991). However, aside from relatively minor differences in ATP binding (Shen et al., 1991), there is no information about other functional differences between the two forms of activase. In this paper we report the effect of elevated temperature on the 45and 41-kD forms of recombinant spinach activase. Our results show that the two forms differ significantly in thermal stability and, when mixed, the thermal stability of the hybrid enzyme is similar to the 45-kD form. The greater thermal stability of the 45-kD form appears to be related to tighter subunit association.