Activation State of Ribulose Bisphosphate Carboxylase in Soybean Leaves

Conditions for extraction and assay of ribulose-1,5-bisphophate carboxylase present in an in vivo active form (initial activity) and an inactive form able to be activated by Mg(2+) and CO(2) (total activity) were examined in leaves of soybean, Glycine max (L.) Merr. cv Will. Total activity was highest after extracts had preincubated in NaHCO(3) (5 millimolar saturating) and Mg(2+) (5 millimolar optimal) for 5 minutes at 25 degrees C or 30 minutes at 0 degrees C before assay. Initial activity was about 70% of total activity. K(act) (Mg(2+)) and K(act) (CO(2)) were approximately 0.3 millimolar and 36 micromolar, respectively. The carry-over of endogenous Mg(2+) in the leaf extract was sufficient to support considerable catalytic activity. While Mg(2+) was essential for both activation and catalysis, Mg(2+) levels greater than 5 millimolar were increasingly inhibitory of catalysis. Similar inhibition by high Mg(2+) was also observed in filtered, centrifuged, or desalted extracts and partially purified enzyme. Activities did not change upon storage of leaves for up to 4 hours in ice water or liquid nitrogen before homogenization, but were about 20% higher in the latter. Activities were also stable for up to 2 hours in leaf extracts stored at 0 degrees C. Initial activity quickly deactivated at 25 degrees C in the absence of high CO(2). Total activity slowly declined irreversibly upon storage of leaf homogenate at 25 degrees C.