PURIFICATION AND CHARACTERIZATION OF ASPARTATE-AMINOTRANSFERASE ISOENZYMES FROM RICE BRAN

Isoenzymes of aspartate aminotransferase (EC 2.6.1.1, AAT) were purified to homogeneity and crystallized from bran of rice (Oryza sativa cv. Koganemasari). The native molecular weights of AAT-1 and AAT-2 isoenzymes were 88,000 and 94,000 with the subunit molecular weights of 44,000 and 47,000, respectively, indicating that the holoenzymes of the isoencymes are dimers. The isoelectric points of AAT-1 and AAT-2 were pH 6.5 and 5.0, respectively: both isoenzymes have no subform. The isoenzymes showed similar K(m)s for four natural substrates, with the exception that AAT-1 had a higher affinity for L-glutamate than AAT-2. Amino donor and acceptor specificities of the isoenzymes were almost identical and fairly high. Amino acid compositions of the isoenzymes were similar but not the same. The isoenzymes contained one mole of pyridoxal 5'-phosphate (PLP) per subunit and showed characteristic absorption spectra of PLP enzymes. Polyclonal antibodies raised against AAT-1 selectively cross-reacted with AAT-1 but not with AAT-2. Conversely, the antibody raised against AAT-2 selectively cross-reacted with AAT-2 but not with AAT-1.