Purification and characterization of anl-amino-acid oxidase fromChlamydomonas reinhardtii

✍ Scribed by P. Piedras; M. Pineda; J. Muñoz; J. Cárdenas

• Publisher: Springer-Verlag
• Year: 1992
• Tongue: English
• Weight: 762 KB
• Volume: 188
• Category: Article
• ISSN: 0032-0935
• DOI: 10.1007/bf00198934

✦ Synopsis

An L-amino-acid oxidase (EC 1.4.3.1) that catalyzes the oxidative deamination of twelve L-amino acids has been purified 21-fold and with 14% yield to electrophoretic homogeneity from Chlamydomonas reinhardtii cells by ammonium-sulfate fractionation, gel filtration through Sephacryl and Superose, anion-exchange chromatography and preparative electrophoresis in polyacrylamide gels. The native enzyme is a protein of 470 kDa and consists of eight identical or similarsized subunits of 60 kDa each. Optimum pH and temperature were 8.2 and 55 ~ C, respectively, with a Q~o (45-55 ~ C) of 1.7 and an activation energy of 45 kJmol-1. Its absorption spectrum showed, in the visible region, maxima at 360 and 444 nm, characteristic of a flavoprotein with a calculated ravin content of 7.7 mol FAD per mol of native enzyme. Apparent Km values of the twelve L-amino acids which can act as substrates of L-amino-acid oxidase ranged between 31 ~M for phenylalanine and 176 gM for methionine. The effect of several specific group reagents, chelating agents and bivalent cations on enzyme activity has also been studied.