PURIFICATION TO HOMOGENEITY AND SOME PROPERTIES OF L-PHENYLALANINE AMMONIA-LYASE OF IRRADIATED MUSTARD (SINAPIS-ALBA L) COTYLEDONS

Lyase (L-Phenylalanine ammonia-lyase, EC 4.3.1.5) from far-red light-irradiated mustard cotyledons was purified to a single protein using ammonium sulfate fractionation, column chromatography on L-phenylalanyl-Sepharose 4B and on Sephadex G-200, islelectric focusing and polyacrylamide gel electrophoresis. The enzyme constituted 0.01% of total cellular protein, did not catalyse the deamination of L-tyrosine, had a pH optimum of pH 8.6 and an isoelectric point of pH 5.6. The sedimentation coefficient was estimated as 11.3 S, the Stokes'' radius 4.25 nm, and the MW 240,000 .+-. 9000 (SE). Electrophoresis on denaturing polyacrylamide gels gave a single stained protein band corresponding to a subunit MW of 55,000, indicating a tetrameric structure of equal (or near-equal) size subunits. Maximum velocity (V) for the purified lyase at 25.degree. C was 3.83-4.10 nkat/l enzyme and the Km value 0.151-0.154 mM. Negative cooperativity (Hill coefficient, n = 1.08) was not detected over the substrate concentration range tested. A putative non-diffusible inhibitor isolated from dark-grown gherkin hypocotyls inhibited the homogeneously purified mustard lyase.