Wound-induced phenylalanine ammonia-lyase in potato (Solanum tuberosum) tuber discs. Significance of glycosylation and immunolocalization of enzyme subunits

Abstract

1. Excised discs of potato (Solanum tuberosum) tuber were incubated with [3H]fucose and extracts were prepared and incubated with an antibody to phenylalanine ammonia-lyase. Analysis of the resulting immunoprecipitated proteins by SDS/PAGE showed [3H]mannose- and [3H]fucose-labelled bands with Mr values corresponding to those of phenylalanine ammonia-lyase subunits. 2. When potato discs were incubated with [3H]sugars in the presence of tunicamycin, an inhibitor of N-linked protein glycosylation, incorporation of radioactivity from [3H]mannose into the immunoprecipitated enzyme subunits was virtually eliminated, whereas that from [3H]fucose was only marginally inhibited. 3. Tunicamycin reduced the level of extractable phenylalanine ammonia-lyase activity induced in excised potato tuber discs. Kinetic analysis revealed that the Vmax value of the enzyme in crude extracts from tunicamycin-treated tissue was reduced, whereas the apparent Km values were unaffected. 4. Immunoprecipitation of the enzyme labelled in vivo with [35S]methionine showed that tunicamycin did not inhibit the synthesis of the enzyme protein per se, nor did it increase the degradation of the enzyme protein. 5. Immunoprecipitation of the enzyme labelled in vitro with [14C]nitromethane showed that tunicamycin did not affect the introduction of the dehydroalanine residue into the active site. 6. These results are consistent with the following hypothesis: tunicamycin inhibits the N-linked glycosylation of phenylalanine ammonia-lyase which, in turn, results in imperfect folding of the enzyme protein. The orientation of the active site is changed in such a way that the affinity of the enzyme for its substrate is unaffected, whereas the catalytic activity of the enzyme is reduced. 7. Both optical- and electron-microscopic immunolocalization studies with antibody to phenylalanine ammonia-lyase showed increased deposition of silver granules in cells in sections of potato discs in which induction of the enzyme was allowed to occur compared with cells from newly wounded tissue. The enzyme was located in the cytoplasm, and was possibly membrane-associated.

Phenylalanine Ammonia-Lyase from Tomato Cell Cultures Inoculated with Verticillium albo-atrum

“…10-40 AM) (14,24). Kinetic parameters have not been reported for tomato PAL, but the enzyme from potato displayed typical complex kinetics (two Km values, 38 and 280 ,uM, and a Hill coefficient of 0.75) (14,24). In contrast with these earlier reports, tomato PAL from inoculated cell cultures displayed normal Michaelis-Menten saturation kinetics and only a single Km value (1 16 AM).…”

“…In either case, the enzyme was purified on the basis of activity, and the sample prepared for SDS-PAGE demonstrated measurable PAL activity. Phenylalanine ammonia-lyase degradation products in the range of 48 kD have also been reported for the enzyme from other species (6,24). The minor high molecular mass band (approximately 100 kD) may represent aggregates of degraded PAL subunits or another contaminating protein.…”

“…>100 AM) and one low (e.g. 10-40 AM) (14,24). Kinetic parameters have not been reported for tomato PAL, but the enzyme from potato displayed typical complex kinetics (two Km values, 38 and 280 ,uM, and a Hill coefficient of 0.75) (14,24).…”

“…The main band migrated as a 74 kD protein, consistent with the molecular mass of PAL purified from other Solanaceous species (reviewed in ref. 14,24). The main contaminating band migrated as a 48 kD protein and probably represents a degradation product of the main PAL subunit.…”

Phenolic Compounds in Maize Grains and Its Nixtamalized Products

“…The biosynthesis of these compounds occurs via the shikimate pathway, from the amino acids phenylalanine and tyrosine and the participation of the enzyme phenylalanine ammonia lyase (PAL) that catalyzes the removal of the ammonia residue of the amino acids phenylalanine and tyrosine to produce cinnamic acid and 4-coumaric acid, respectively [5]. Both compounds further enter the phenylpropanoid pathway and it is within the various branches of this pathway where the great diversity of phenolic compounds so far identiied is synthesized.…”