Wound-induced phenylalanine ammonia-lyase in potato (Solanum tuberosum) tuber discs. Significance of glycosylation and immunolocalization of enzyme subunits
- 1 April 1990
- journal article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 267 (1), 163-170
- https://doi.org/10.1042/bj2670163
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.Keywords
This publication has 20 references indexed in Scilit:
- Hypoxic Stress Inhibits the Appearance of Wound-Response Proteins in Potato TubersPlant Physiology, 1988
- Protein Degradation in Lemna with Particular Reference to Ribulose Bisphosphate CarboxylasePlant Physiology, 1987
- Oligosaccharide Side Chains of Glycoproteins that Remain in the High-Mannose Form Are Not Accessible to GlycosidasesPlant Physiology, 1986
- Use of nitrogen-15 and deuterium isotope effects to determine the chemical mechanism of phenylalanine ammonia-lyaseBiochemistry, 1985
- Fucosylation of Membrane Proteins in Soybean Cultured CellsPlant Physiology, 1985
- Tunicamycin Inhibits Protein Glycosylation in Suspension Cultured Soybean CellsPlant Physiology, 1981
- Effects of Gibberellic Acid and of Tunicamycin on Glycosyl‐Transferase Activities and on α‐Amylase Secretion in BarleyEuropean Journal of Biochemistry, 1979
- A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye BindingAnalytical Biochemistry, 1976
- L-Phenylalanine ammonia-lyase (maize, potato, and Rhodotorula glutinis). Studies of the prosthetic group with nitromethaneBiochemistry, 1975
- L-Phenylalanine ammonia-lyase (maize and potato). Evidence that the enzyme is composed of four subunitsBiochemistry, 1973