Determination of Apparent Km Values for Ribulose 15-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Using the Spectrophotometric Assay of Rubisco Activity

摘要

The spectrophotometric assay for ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was used to determine the rate of increase in Rubisco activity over time in the presence or absence of Rubisco activase. Polynomial approximations to the raw data were used to smooth out minor fluctuations in the spectrophotometer readings, and Rubisco activase activity was expressed as nanomoles of activated Rubisco per minute. This assay was used to examine the effects of CO2 and the inactive-Rubisco:ribulose 1,5-bisphosphate complex (ER) on the activase-catalyzed activation reaction. Double-reciprocal plots of activase activity and ER at several concentrations of CO2 were consistent with two-substrate Michaelis-Menton kinetics, and the apparent Km (CO2) and Km(ER) were determined to be 53 and 2.7 micromolar, respectively. These data do not prove that ER and CO2 are substrates for the reaction catalyzed by activase, but they may be important to our understanding of the activation process in vivo. The implications of these data and their relation to previously published data on the effects of ER and CO2 on activase are discussed.