Purification and Characterization of Rubisco in Porphyra okamurae Ueda

Algae > Volume 15(3); 2000 > Article

Algae 2000;15(3): 155-162.

Jae-Suk Choi, Ik Kyo Chung, Bang-Ook Jun

ABSTRACT

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) was extracted and purified from the fresh thalli of Porphyra okamurae Ueda, and the enzymatic properties were investigated. The isolation procedure for purifying rubisco involved precipitation with 18% (w/v) PEG and 200 mM Mg supper(2+) then ion exchange chromatography on Toyopearl DEAE-650 and gel chromatography on Sephadex G-200. The purified rubisco was partially contaminated by phycobiliprotein. The final purification factor was 6.80 and the specific activity was 125.13 nmole CO sub(2)/mg protein/min. The native molecular weight of the purified rubisco was estimated to be 540 kD by Sephacryl S-300 gel filtration chromatography. The enzyme was thought to have a conventional L sub(8) S sub(8) composition. The molecular weight of large subunit and small subunit were 54.0 kD and 16.7 kD, respectively. The enzyme showed a maximum activity at 20 mM Mg supper(2+), 20 mM NaHCO sub(3) and 2.5 mM RuBP. The apparent Km values for NaHCO sub(3) and RuBP were 3.65 mM and 1.2 mM respectively. These data provided some fundamental knowledge to seaweed rubisco that had not been studied thoroughly, and were compared with those from terrestrial plants and other algae to stress the unique features of rhodophytic rubisco.