Purification to homogeneity and some properties of L-phenylalanine ammonia-lyase of irradiated mustard (Sinapis alba L.) cotyledons.

1. 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 sulphate fractionation, column chromatography on L-phenylalanyl-Sepharose 4B and on Sephadex G-200, isoelectric focusing and polyacryalmide gel electrophoresis. 2. 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. 3. The sedimentation coefficient was estimated as 11.3 S, the Stokes' radius 4.25 nm, and the molecular weight 240 000 +/- 9000 (S.E.). 4. Electrophoresis on denaturing polyacrylamide gels gave a single stained protein band corresponding to a subunit molecular weight of 55 000 indicating a tetrameric structure of equal (or near-equal) size subunits. 5. Maximum velocity (V) for the purified lyase at 25 degrees C was 3.83--4.10 nkat. 1(-1) enzyme and Km value 0.151--0.154 mM. Negative cooperativity (Hill coefficient, n = 1.08) was not detected over the substrate concentration range tested. 6. A putative non-diffusible inhibitor isolated from dark-grown gherkin hypocotyls inhibited the homogeneously purified mustard lyase.