Detoxification of N-(phosphonoacetyl)-L-aspartate by carrot cells in suspension culture

Abstract

In bacterial and mammalian cells, N-(phosphonoacetyl)-L-aspartate (PALA) suppresses growth by strongly inhibiting aspartate transcarbamoylase (ATCase; EC 2.1.3.2), a key enzyme of the pyrimidine biosynthetic pathway. At a concentration that would suppress growth in mammalian or bacterial cells, and that is nearly a million-fold greater than the inhibition constant (K i ) for ATCase in carrot (Daucus carota) seedling extracts, PALA does not suppress growth of carrot cells in suspension culture. To study this anomaly an assay based on the inhibition of wheat (Triticum vulgare) ATCase (K i =2 nM) was developed. Using this assay it was found that PALA is detoxified relatively rapidly by low inocula of carrot cells. The detoxification product accumulates in the extracellular fluid although the enzyme(s) responsible is intracellular or in the cell wall. The PALA-detoxifying activity can be detected at all stages of the growth cycle in culture, but reaches a maximum early in the exponential phase of growth. Cells that were repeatedly subcultured into media initially containing 1 mM PALA had the same low level of ATCase activity as control cells; there was no evidence of the amplification of the gene for this enzyme, such as occurs in mammalian cells upon repeated exposure to the drug.