Limited Proteolysis of Barley Rubisco and Recognition of the Fragments by Monoclonal Antibodies

Abstract

Little is known about the fragmentation of Rubisco by endogenous and exogenous proteases. More sensitive to proteolysis is Rubisco large subunit (LS) and the characteristic pattern of in vivo degrading fragments from Rubisco is different depending on experimental conditions (5, 7, 9, 10). Under limited proteolysis of native spinach Rubisco with trypsin two short peptides were released from the N-terminus and one from the C-terminus of the LS (13). Denatured Rubisco from spinach is digested by different proteases (chymotrypsin, papain and St. aureus V8 protease) and the LS of the enzyme generates about 15–25 bands (1). Barley Rubisco is less sensitive to trypsinolysis compared to wheat and spinach Rubisco (6). The characterization of Mabs against Rubisco applying limited proteolysis of the enzyme has not been made so far. Monoclonal antibodies (Mabs) against Rubisco from barley leaves have been produced and partially characterized (11). All the Mabs recognise only LS in Western blotting. In the present paper an attempt was made to more thoroughly characterize the epitopes on Rubisco molecule recognized by the Mabs, using limited proteolysis.