RIBULOSE BISPHOSPHATE CARBOXYLASES FROM MACROALGAE - PROTEOLYSIS DURING EXTRACTION AND PROPERTIES OF THE ENZYME FROM PORPHYRA-UMBILICALIS

The ribulose-1,5-bisphosphate carboxylase/oxygenase present in six macroalgae from the Rhodophyceae and Chlorophyceae was assayed, and the enzyme purified from Porphyra umbilicalis. In crude extracts from this red alga the carboxylase activity was typically 19 nmol CO2 fixed min-1 mg-1 protein, and the oxygenase activity 8 nmol O2 utilised min-1 mg-1 protein. In cell extracts the enzyme existed in two molecular forms which differed sufficiently in M(r) to be separable on PAGE. On SDS-PAGE two forms of the large subunit of the enzyme were identified. One of these originated by protease action during cell disruption as inclusion of protease inhibitors in the extraction medium eliminated the heterogeneity. The enzyme could be purified by a two-step procedure based on FPLC; the highest specific activity obtained for the isolated enzyme was 100 nmol CO2 fixed min-1 mg-1 protein. The ribulose bisphosphate carboxylase/oxygenase from Porphyra umbilicalis had a sedimentation coefficient (s20-degrees, w) of 19.2 x 10(-13) sec and a diffusion coefficient (D20-degrees, w) of 3.2 x 10(-7) cm2 sec-1 giving a M(r) of 560 000 by the Svedberg equation; the M(r) sedimentation equilibrium was 525 000. Subunits of M(r) 53 000 and 16 000 were demonstrated by SDS-PAGE suggesting the enzyme had a conventional L8S8 composition.