Waltham M C, Lin S, Li W W, Göker E, Gritsman H, Tong W P, Bertino J R
Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
J Chromatogr B Biomed Sci Appl. 1997 Feb 21;689(2):387-92. doi: 10.1016/s0378-4347(96)00350-7.
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.
本文描述了一种使用毛细管电泳（CE）分离甲氨蝶呤（MTX）多聚谷氨酸盐的快速灵敏方法，该方法适用于体外检测γ-谷氨酰水解酶（GGH，EC 3.4.22.12）。使用内径75微米的毛细管（50厘米，+25千伏），可在10分钟内实现MTX及其多聚谷氨酸化形式（直至glu4）的清晰分离，并能够对反应物和酶产物进行定量分析。由于使用少于5×10⁵个真核细胞就能可靠地测定活性，这项新技术可用于测量患者肿瘤样本中的GGH，并研究GGH水平与临床MTX耐药性之间的关系。
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