[Separation of methotrexate-polyglutamates by capillary electrophoresis and its application to the measurement of gamma-glutamyl hydrolase activity in human leukemia cells in culture].
We have applied capillary electrophoresis to the separation of methotrexate (MTX)-polyglutamates, and gamma-glutamyl hydrolase (GGH) activities in tumor cells were measured by using this new analytical method. MTX-polyglutamates were sufficiently separated in 15min by capillary electrophoresis with silica fused capillary (phi 50 microns x 75cm), being electrophoresed at 25kV and 30 degrees C in a buffer which contained 20mM sodium tetraborate, 20mM SDS and adjusted pH to 9.5. MTX-polyglutamates eluted were detected at 300nm UV. Cellular extracts obtained from the sensitive and antifolate-resistant human leukemia cell lines, MOLT-3 and K562, were incubated with MTX-glu5 at 37 degrees C for 1, 2 and 4 hr, and the amounts of the degradation products (glu1-glu4) were measured for GGH activity by capillary electrophoresis. There was no significant difference in the production of the metabolites between MOLT-3 and K562 cells (867 +/- 109 vs 799 +/- 56 pmol products/min/1 x 10(7) cells), however, the MTX-resistant MOLT-3 cells with a diminished polyglutamation of folates (MOLT-3/MTX.P-17) and the ZD1694-resistant K562 cells with the impaired membrane transport for reduced folates/MTX/ZD1694 (K562/ZD1694.C) showed decreased activities of GGH (519 +/- 52 and 680 +/- 99 pmol products/min/1 x 10(7) cells, respectively), suggesting the down-regulation of the enzyme in these antifolate-resistant cells concomitant with the intracellular substrate depletion. This study indicates that capillary electrophoresis is a rapid, cost-efficacious method with a sufficient reproducibility in the measurement of GGH activity and must be more suitable for the analysis of clinical samples than HPLC method which requires a large volume of the material.
Takemura
Y
Y
Department of Laboratory Medicine, National Defense Medical College, Tokorozawa, Saitama, Japan.
Kobayashi
H
H
Sekiguchi
S
S
D015536:Down-Regulation; D019075:Electrophoresis, Capillary; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000201:enzymology*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000302:isolation & purification; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000302:isolation & purification; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology*; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured / Q000201:enzymology; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism*
D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D011623:gamma-Glutamyl Hydrolase; D008727:Methotrexate