PLANT PHYSIOLOGY , Vol 104, Issue 2 373-380, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Folate Metabolism in Datura innoxia (In Vivo and in Vitro Folylpolyglutamate Synthesis in Wild-Type and Methotrexate-Resistant Cells)
K. Wu, E. A. Cossins and J. King
Department of Biology, University of Saskatchewan, Saskatoon, Canada S7N 0W0 (K.W., J.K.)
In vivo folylpolyglutamate pools of the wild-type (Px4) and
methotrexate-resistant (MTX161) Datura innoxia cell lines were detected by
incorporation of [14C]p-aminobenzoate into folates. The folylpolyglutamate
derivatives were cleaved to p-aminobenzoylpolyglutamates and separated
according to glutamyl chain length by high-performance liquid
chromatography. Hexaglutamates were the predominant form in both Datura
cell lines. The proportions of individual folylpolyglutamates were
unaffected by culturing the cells in medium containing products of
one-carbon metabolism such as glycine, adenine, thymidine, or methionine.
Radiolabeling of the hexaglutamates was greatly reduced in the presence of
10-8 M methotrexate (MTX) in the Px4 cells but not in the MTX161 cells.
Tetrahydrofolate, 5, 10-methylenetetrahydrofolate, and folinic acid were
effective substrates for the folylpolyglutamate synthetase from Datura
cells in vitro, whereas MTX and folate were poor substrates. In vivo, MTX
can be slowly converted into its polyglutamate derivatives up to MTXGlu4 or
MTXGlu5 in Datura cells in the longer term. Significantly lower levels of
MTX polyglutamates in MTX161 cells were found compared with those of Px4
cells during prolonged (10 d) exposure to MTX. Although in vivo and in
vitro folylpolyglutamate synthesis was found to be similar in both cell
lines, about a 4-fold increase in specific activity of [gamma]-glutamyl
hydrolase (GGH) was detected in the MTX161 cell line. The increase in GGH
in the resistant cells suggested that breakdown of polyglutamylated forms
of MTX may play a role in acquired MTX resistance.
