Purified Ribulose-P2 carboxylase from wheat with high specific activity and with fast activation

Abstract

Isolation of Ribulose-P2 carboxylase/oxygenase and its purification to homogeneity often results in losses of enzyme activity. Attempts were made to decrease these losses by adding fructose-P2, SO ]-, Pi or PPi to the buffers used during early stages of the extraction and purification of the enzyme. Yield and final specific activity were not improved, but fructose-P2 and PPi became bound to the enzyme and modified its activation characteristics. The enzyme prepared with fructose-P2 showed a much decreased rate of deactivation when the activating cofactors Mg 2÷ or CO2 were reduced in concentration. Specific activities of above 1.9 pmol CO2 min -1 mg -1 protein were found at intermediate stages in the purifications. High specific activities from all preparations require extraction from preilluminated young leaves of plants given excess N. Decrease in specific activity in the final stages of purification was not generally seen if Pi was added during activation of fractions with Mg a ÷ and CO2 prior to assay.Differences in tertiary and quaternary structure of the various preparations were examined using polyacrylamide gel electrophoresis and by a preliminary study of the growth and properties of crystals. Similar crystals were obtained from them all but gel electrophoresis showed that preparations made in the presence of fructose-Pz contained a greater proportion of aggregated states of ribulose-P2 carboxylase/oxygenase subunits giving protein of M.Wt. greater than 5 50,000 Daltons.

Nitrate Nutrition and Temperature Effects on Wheat: Enzyme Composition, Nitrate and Total Amino Acid Content of Leaves

“…RuBPc-o activity was proportional to the amount of protein, so that the mean specific activity was 3-74 /imol g RuBPc-o protein" l s" 1 (Table 1) corresponding to approximately 6-4 fimo\ g RuBPc-o protein" 1 s" 1 at 25 °C. This was lower than the 30 fxmo\ g RuBPc-o protein" 1 s" 1 determined by Schmidt, Cornelius, Burton, Parry, Millard, Keys, and Gutteridge (1984) for purified protein under optimal conditions. Makino et al (1984) measured specific activity of 80 fimo\ g RuBPc-o protein" 1 s" 1 in rice.…”

Nitrate Nutrition and Temperature Effects on Wheat: Photosynthesis and Photorespiration of Leaves

“…RuBPc-o may have low catalytic activity in vitro due to inadequate CO2 or magnesium for activation, or to incorrect pH (Boyle, 1983) or to decreased availability of inorganic phosphate and increased concentrations of non-reducing sugars, sugar phosphates etc. (Friedrich and Huffaker, 1980;Ku, Prickril, Reger, and Pallas, 1982;Schmidt, Cornelius, Burton, Parry, Millard, Keys, and Gutteridge, 1984) but the importance of these factors in vivo has not been assessed. Products of the PCR cycle might be expected to accumulate in chloroplasts with high rates of />", inhibiting assimilation.…”