gamma-Glutamyl hydrolase from human sarcoma HT-1080 cells: characterization and inhibition by glutamine antagonists.
gamma-Glutamyl hydrolase from human sarcoma HT-1080 cells: characterization and inhibition by glutamine antagonists.
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DOI：
10.1124/mol.51.5.825
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发表时间：
1997-05
影响因子：
3.6
通讯作者：
M. Waltham;W. Li;H. Gritsman;W. Tong;J. Bertino
中科院分区：
文献类型：
--
作者：
M. Waltham;W. Li;H. Gritsman;W. Tong;J. Bertino
高γ -谷氨酰水解酶（GGH）活性作为一个促进因素在机制获得性和内在抗叶酸抗性已经报道了几个培养细胞系。尽管如此，人们对这种酶知之甚少，尤其是人类。利用人HT-1080肉瘤细胞系，我们观察到GGH活性在培养过程中分泌到培养基中（这种现象可以通过暴露于NH4Cl显著刺激），并且酸性pH值最适合酶的体外催化活性。这些特性与酶的溶酶体位置一致。与啮齿类动物的GGH不同，HT-1080酶（纯化的<或= 2000倍）的制剂显示出从叶酸、甲氨蝶呤（MTX）和对氨基苯甲酸底物的多l - γ -谷氨酰衍生物中切割连续末端-谷氨酰基团的外肽酶活性，并明显倾向于长链多谷氨酸（glu4衍生物的Km值比glu1衍生物的Km值分别低17倍和15倍）。通过体外实验筛选，几种谷氨酰胺拮抗剂[即6-重氮-5-氧-去甲亮氨酸（DON）， acivicin和azaserine]被鉴定为人类GGH抑制剂，其中DON是最有效的，并表现出时间依赖性的抑制作用。在细胞培养实验中，同时暴露于DON（10微米）和[3H]MTX 24小时可导致HT-1080和另一种人肉瘤系抗叶酸长链γ -谷氨酰衍生物的适度升高。这些化合物可以作为有用的先导化合物，用于开发特定的GGH抑制剂，用于检查GGH活性与抗叶酸作用之间的关系，并可能在临床中与需要多谷氨酰化才能有效保留细胞的抗叶酸药物联合使用。
Elevated gamma-glutamyl hydrolase (GGH) activity as a contributing factor in mechanisms of acquired and intrinsic antifolate resistance has been reported for several cultured cell lines. Despite this, little is known about this enzyme, especially the human species. Using the human HT-1080 sarcoma line, we observed the secretion of GGH activity into media during culture (a phenomenon that could be markedly stimulated by exposure to NH4Cl) and an acidic pH optimum for in vitro catalytic activity of the enzyme. These properties are consistent with a lysosomal location for the enzyme. Unlike rodent GGH, preparations of HT-1080 enzyme (purified < or = 2000-fold) displayed exopeptidase activity in cleaving successive end-terminal gamma-glutamyl groups from poly-L-gamma-glutamyl derivatives of folate, methotrexate (MTX), and para-aminobenzoic acid substrates and a marked preference for long-chain polyglutamates (Km values for glu4 versus glu1 derivatives were 17- and 15-fold lower for folate and MTX versions, respectively). Using an in vitro assay screen, several glutamine antagonists [i.e., 6-diazo-5-oxo-norleucine (DON), acivicin, and azaserine] were identified as human GGH inhibitors, with DON being the most potent and displaying time-dependent inhibition. In cell culture experiments, simultaneous exposure of DON (10 microM) and [3H]MTX for 24 hr resulted in modest elevations of the long-chain gamma-glutamyl derivatives of the antifolate for HT-1080 and another human sarcoma line. These compounds may serve as useful lead compounds in the development of specific GGH inhibitors for use in examining the relationship between GGH activity and antifolate action and may potentially be used in clinical combination with antifolates that require polyglutamylation for effective cellular retention.