Phenylalanine ammonia-lyase from illuminated buckwheat hypocotyls and production of monoclonal antibodies

Abstract

Phenylalanine ammonia-lyase (PAL) was purified to apparent homogeneity from illuminated buckwheat hypocotyls. The purification process involved ammonium sulphate fractionation, gel filtration, ion-exchange chromatography on DEAE-Bio-Gel, and chromatofocusing on PBE 94. The purified enzyme had a M r of 330 000 ± 16 500 with a subunit size of M r 85 000 ± 4000. Optimal activity of the enzyme was observed at pH 8.8. An apparent K m value of 157 μM was determined for phenylalanine which was the only substrate utilized by the enzyme. Antibodies against the buckwheat PAL were developed using standard monoclonal techniques. A preparation containing antibodies from three monoclonal lines was able to precipitate 100% of the enzyme activity from crude buckwheat extracts. Western blots showed immunorecognition of proteins with M r of 70 000; 65 000; and 55 000 in addition to the PAL subunit of M r 85 000. These additional proteins appeared to be degradation products of PAL.