Purification, characterization and thermostability of ribulose 1,5-bisphosphate carboxylase-oxygenase from barley leaves

The enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) and it s functional subunits from barley (Hordeum vulgare L.) leaves were purified to homogeneity by activity-directed sequencial steps of chromatography. Ba sed on the molecular mass estimation by SDS-PAGE, the large subunit (LS) ha d an apparent molecular weight of cn. 55 kDa, whereas the small subunit (SS ) was ca. a 14 kDa polypeptide chain. The N-terminal sequences, established by automated Edman degradation analysis of the purified subunits, showed v ery close sequence homologies (52-92%) with the subunits of other rubisco e nzymes reported from several photosynthetic species. In order to establish the chemical heterogeneity in the rubisco from barley, the amino acid compo sition of purified native enzyme was analyzed and the results systematicall y compared with other known type-I rubisco enzymes from spinach, maize, tob acco and pea. Major differences have been observed in the amino acid compos ition of barley rubisco, the concentration of cysteine, serine, threonine, isoleucine, leucine, arginine and tryptophan residues were found quite vari able as compared to other higher plants. The thermostability of the native rubisco was also investigated using circular dichroism and fluorescence spe ctroscopy. The critical (T-c) and melting (T-m) temperatures were determine d to be 60 degrees C and 57 degrees C respectively, and at this temperature the enzyme not only retains its structural integrity but also its enzymati c activity. Results of these studies were discussed in the light of structu ral and functional adaptation of this bifunctional enzyme in C-3 and C-4 pl ants to their environments.