Chacon, Rosaura Maria Romero; (1994) Enzymes of chorismate metabolism in ailanthus altissima (mill.) swingle cell cultures. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The thesis incorporates the results of an investigation of chorismate utilising enzymes in Ailanthus altissima (Mill.) Swingle cell cultures. These enzymes are anthranilate synthase, chorismate mutase and isochorismate synthase. The activity of the three enzymes was measured in callus and cell suspension cultures of the plant, but only anthranilate synthase (AS) and chorismate mutase (CM) were detected. A time course study was carried out to determine the growth and the enzymes' activities. The study showed that AS measured after use of a Sephadex PD-10 column, was active through the cell growth cycle, giving a maximum activity within 11 and 17 days, whereas CM activity increased after 23 days. CM activity was 150x that of AS. Two AS enzymes were isolated from callus and cell suspension cultures of the same cell line. The enzymes were purified by 10% and 18% polyethylene glycol precipitation (PEG) followed by separation on a Q-Sepharose (Pharmacia) column using FPLC with a discontinuous NaCl gradient. Further protein purification was achieved with a Mono Q column (Pharmacia) with discontinuous NaCl gradient to yield both isoenzymes. The two isoenzymes showed different regulation by tryptophan. One of the AS enzyme present was characterised. The molecular Weight of the enzyme using gel filtration was 77±3 kDa, and the Km respect to the substrates L-glutamine and chorismate, and the cofactor Mg2+ were determined as 0.050mM, 72μM and 0.18mM, respectively. An optimum pH between 7.3 and 8, and an optimum temperature of 35°C were also determined. Two CM enzymes were also detected, but they were not characterised. The activities and regulations of AS and CM enzymes are discussed and compared with other data from higher plants and microorganisms.

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