L-Phenylalanine ammonia-lyase from Phaseolus vulgaris: partial degradation of enzyme subunits in vitro and in vivo

Abstract

L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified from suspension cultured cells of French bean ( Phaseolus vulgaris L.) which had been exposed to polysaccharide elicitor preparations from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. After preliminary purification by ammonium sulphate fractionation and gel filtration, the enzyme was further purified by (a) ion-exchange chromatography followed by chromatofocussing, (b) chromatography on rabbit anti-(phenylalanine ammonia-lyase) IgG, or (c) affinity chromatography on L-aminooxy( p-hydroxyphenyl)propionic acid (or L-tyrosine) linked to epoxy-activated Sepharose 6B via the phenolic hydroxyl group. The purified enzyme preparations exhibited subunit M r values of 77 000, 70 000 and 53 000, the relative proportions of these depending upon the enzyme source, length of time taken for purification, and inclusion of freeze-thaw steps. Four forms of the enzyme, differing in p I value, were resolved by chromatofocussing, although all forms from the same preparation consisted of similar proportions of the different subunit M r forms. Peptide mapping and freeze-thaw studies indicate that the M r 77 000 native phenylalanine ammonia-lyase subunit is inherently unstable in vitro and breaks down to yield the lower M r partial degradation products. Such products could also be observed following in vitro translation of phenylalanine ammonia-lyase mRNA. Pulse-chase experiments indicated that the 77 000 → 70 000 → 53 000 subunit interconversion also occurs in vivo.