Assay of isochorismate synthase from plant cell cultures by high-performance liquid chromatography
1991
Poulsen, C. | Heijden, R. van der | Verpoorte, R.
An HPLC assay is described for the enzyme isochorismate synthase (EC 5.4.99.6) in which the formation of isochorismate and the utilization of chorismate is followed at the same time. The assay shows superior linearity with respect to enzyme activity compared to the presently used fluorometric assay. The detection limit with the present HPLC assay is 1.8 ng or 5 pmol isochorismate. With the HPLC assay the specific activity of isochorismate synthase in Rubia tinctorum, R. tinctorum 'hairy root', and Morinda citrifolia cell cultures were measured. Of these cell lines R. tinctorum suspension cells, five days after inoculation, had the highest specific activity, 2.2 pkat mg(-1) protein. The enzyme activity was found in an anthraquinone-producing M. citrifolia cell line, but not in a non-producing cell line, pointing to the significance of this enzyme in the regulation of anthraquinone production. Plant isochorismate synthase shows an absolute Mg(2+) requirement. The enzyme is stabilized by glycerol, EDTA, and DTT. Protease inhibitors had no effect on the stability. Isochorismate synthase from R. tinctorum is stable at -80 degrees for at least two months.
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