Characterization of the regulatory function of the 46-kDa isoform of Rubisco activase from Arabidopsis

Arabidopsis Rubisco activase was recently shown to be regulated by redox ch anges in the larger (46-kDa) isoform specifically mediated by thioredoxin-f [Zhang and Portis (1999) Proc Natl Acad Sci USA 96: 9438-9443]. Reduction greatly increases the activity of the 46-kDa isoform and the native protein at physiological ATP/ADP ratios. In this study we conducted additional exp eriments to characterize the regulation of Rubisco activase by thioredoxin- f. The K m for both ATP hydrolysis and Rubisco activation by the 46-kDa iso form was lowered by 4 to 5-fold after reduction, but the maximum activity w as increased by only 10%. Only 0.35 muM thioredoxin-f was required for a ha lf-maximal activity change after a 10 min preincubation and activation with 1 muM was complete after 10 min. Equal amounts of 46-kDa and 43-kDa isofor ms were required for a complete inhibition of the Rubisco activation activi ty after a reduction-oxidation cycle and assay at an ATP/ADP ratio of 3:1, whereas activity was only inhibited by 50% at a 2:1 ratio (43-/46-kDa) of t he isoforms. This requirement is consistent with the fact that Arabidopsis normally contains about a 1:1 ratio of the two isoforms at both the mRNA an d protein levels. Redox titrations indicated a midpoint potential of -344 m V for the 46-kDa isoform as compared to -342 mV for spinach fructose 1,6-bi sphosphatase at pH 7.9, consistent with previous reports indicating that th ese proteins are co-regulated by light intensity in a similar manner.