Purification, characterization and induction of L-phenylalanine ammonia-lyase in Phaseolus vulgaris.
The enzyme L-phenylalanine ammonia-lyase was purified from leaves of Phaseolus vulgaris by Sephacryl S-200 gel filtration and Sepharose-4-B--succinyl-aminoethyl-L-phenylalanine affinity chromatography. L-Phenylalanine ammonia-lyase was specifically eluted from the affinity matrix with its substrate L-phenylalanine at 20-25 degrees C. The purified enzyme was shown to be homogeneous by gel electrophoresis both in presence and absence of SDS. Its Mr, determined by gel filtration and non-denaturing gel electrophoresis, was 320,000 +/- 9000 and 330,000 +/- 4000 respectively. After SDS electrophoresis only one band of Mr 83,000 +/- 4000 was detected, indicating that the enzyme is an oligomer containing four subunits. The pH optimum of enzyme activity was 8.8-9.2. Ampholyte isoelectrofocusing in polyacrylamide demonstrated the presence of a single charged species at pH 4.2. The homogeneous enzyme catalyzed the deamination of L-phenylalanine to trans-cinnamate but did not catalyze the transamination of L-phenylalanine to L-phenylpyruvate. The enzyme showed Km 1.25 mM for L-phenylalanine. Antibodies to homogeneous L-phenylalanine ammonia-lyase recognised specific epitopes on L-phenylalanine aminotransferase as demonstrated by immunoaffinity purification and immunoblotting. The induction of L-phenylalanine ammonia-lyase activity during phaseollin biosynthesis in the Phaseolus vulgaris--Colletotrichum lindemuthianum interaction was regulated by an increase in enzyme concentration resulting from an increase in de novo synthesis of L-phenylalanine ammonia-lyase protein.
da Cunha
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Department of Plant Sciences, University of Oxford.
D000642:Ammonia-Lyases / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D007525:Isoelectric Focusing; D010649:Phenylalanine / Q000494:pharmacology; D010650:Phenylalanine Ammonia-Lyase / Q000302:isolation & purification*; D010940:Plant Proteins / Q000096:biosynthesis; D010944:Plants / Q000201:enzymology* / Q000378:metabolism; D000637:Transaminases / Q000302:isolation & purification
D010940:Plant Proteins; D010649:Phenylalanine; D000637:Transaminases; C022340:phenylalanine aminotransferase; D000642:Ammonia-Lyases; D010650:Phenylalanine Ammonia-Lyase