Article

Purification and characterization of phenylalanine ammonia-lyase from Ustilago maydis

Department of Microbiology and Immunology, University of British Columbia - Vancouver, Vancouver, British Columbia, Canada
Phytochemistry (Impact Factor: 3.35). 09/1996; 43(2):351-357. DOI: 10.1016/0031-9422(96)00282-8

ABSTRACT Phenylalanine ammonia-lyase (PAL; EC. 4.3.1.5) has been purified to homogeneity from liquid-cultured cells of the phytopathogenic fungus Ustilago maydis by use of heat treatment, protamine and ammonium sulphate precipitation, ion-exchange and gel filtration chromatography, and preparative PAGE. Its native molecular mass was estimated as 320±20 kDa and its subunit molecular mass as 80 kDa. No isoforms of the enzyme were detected, and there was no evidence of glycosylation of the protein. Ustilago PAL was most active at pH 8.8–9.2 and 30° and had a Km for l-phenylalanine of 1.05 mM. The enzyme did not deaminate l-tyrosine. The synthetic inhibitor 2-aminoindan-2-phosphonic acid (AIP) strongly inhibited the enzyme, as did sulphhydryl reagents and carbonyl reagents, whereas t-cinnamate was only moderately inhibitory. Ustilago PAL activity had no requirement for metal ion cofactors, but was inhibited by heavy metal ions (Ag+, Cu2+, and Hg2+). Polyclonal antibodies raised against the purified enzyme readily recognized U. maydis PAL in solution and on Western blots, but only weakly cross-reacted with higher plant PAL.

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