Capillary electrophoresis of methotrexate polyglutamates and its application in evaluation of gamma-glutamyl hydrolase activity.
A rapid and sensitive procedure for the separation of methotrexate (MTX) polyglutamates2 using capillary electrophoresis (CE) is described as it applies to the in vitro assay of the enzyme gamma-glutamyl hydrolase (GGH, EC 3.4.22.12). Distinct separation of MTX and polyglutamylated forms (up to glu4) is achieved within 10 min using a 75 microns I.D. capillary (50 cm, +25 kV), and enables quantitation of both reactant and enzyme products. As activity can be reliably determined using less than 5 x 10(5) eukaryotic cells, this new technique can be used to measure GGH in patient tumor samples and investigate the relationship between GGH levels and clinical MTX resistance.
Waltham
M C
MC
Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Lin
S
S
Li
W W
WW
Göker
E
E
Gritsman
H
H
Tong
W P
WP
Bertino
J R
JR
D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000032:analysis; D015394:Molecular Structure; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000032:analysis; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism*
D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D011623:gamma-Glutamyl Hydrolase; D008727:Methotrexate