Purification and characterization of large and small subunits of ribulose 1,5-bisphosphate carboxylase expressed separately in Escherichia coli

Procedures were developed for 95 and 80% purification to homogeneity of the large subunit (L) and small subunit (S) of ribulose 1,5-bisphosphate carboxylase/oxygenase (L 8S 8) from Synechococcus PCC 6301, each expressed separately in Escherichia coli. Purified L had a low specific activity in the absence of S (0.075 μmol CO 2 fixed/ mg holoenzyme/min). Following elution on a Pharmacia Superose 6 or 12 gel filtration column, 50% of the purified L appeared as the octamer, L 8. The rest was in equilibrium with lower polymeric species and/or was retained on the column. Large and small subunits assembled rapidly into the L 8S 8 holoenzyme that had high specific activities, 6.2 and 3.1 μmol CO 2 fixed/mg holoenzyme/min for the homologous Synechococcus L 8S 8 and the hybrid Synechococcus L-pea S L 8S 8, respectively. The CO 2 dependence for carbamylation of L 8 was compared to that of L 8S 8 as a function of pH and CO 2 concentration. The pH dependence indicated an apparent p K a for L 8 of 8.28 and for L 8S 8 of 8.15, suggesting that S may influence the p K a of the lysine involved in carbamylation. The K act for CO 2 at pH 8.4 were similar for L 8 (13.5 μ m) and L 8S 8 (15.5 μ m). L 8 bound 2-[ 14C]carboxy- d-arabinitol 1,5-bisphosphate (CABP) tightly so that most of the bound [ 14C]CABP survived gel filtration. A major amount of the L 8-[ 14C]CABP complex appeared as larger polymeric aggregates when eluted in the presence of E. coli protein.