Yao R, Rhee M S, Galivan J
Wadsworth Center, New York State Department of Health, Albany 12201-0509, USA.
Mol Pharmacol. 1995 Sep;48(3):505-11.
A subline of H35 hepatoma cells (H35D cells) that have been made resistant to 5,10-dideazatetrahydrofolate exhibits an increase in gamma-glutamyl hydrolase (GH) activity. GH is a lysosomal enzyme in H35 and H35D cells on the basis of comparison of the distribution of enzyme activity with other known lysosomal enzymes. The hydrolysis rate of methotrexate polyglutamate with isolated, intact lysosomes is 4-5-fold greater in H35D cells than in H35 cells. GH activity in isolated lysosomes is in part dependent on the presence of a reducing agent such as mercaptoethanol. Permeabilization of lysosomal preparations from both cell types by Triton X-100 causes a 10-fold enhancement in GH activity. The result of the enhanced activity of GH in H35D cells is a marked reduction in antifolylpolyglutamate concentration, with the parent antifolate being the predominant intracellular species found under all conditions tested. Unlike antifolates, the total intracellular folate concentration is nearly identical in both cells under standard culture conditions up to 10 microns folic acid. However, the chain length of folylpolyglutamates consists of predominantly triglutamates and tetraglutamates in H35D cells with increased GH, whereas it consists of pentaglutamates and hexaglutamates in the parental cells. At 50 and 100 microns folic acid, the folate accumulation in H35D cells is less than half that of H35 cells, and the predominant polyglutamate species in the H35D cells are the diglutamates through the tetraglutamates. The results demonstrate that the two H35 cell lines having equal folylpolyglutamate synthetase but that one with enhanced lysosomal GH activity exhibits a marked reduction in the amount and gamma-glutamyl chain length of folylpolyglutamates and antifolylpolyglutamates.
一种对5,10-二去氮四氢叶酸产生抗性的H35肝癌细胞亚系（H35D细胞），其γ-谷氨酰水解酶（GH）活性有所增加。基于酶活性分布与其他已知溶酶体酶的比较，GH在H35和H35D细胞中是一种溶酶体酶。在分离的完整溶酶体中，甲氨蝶呤多聚谷氨酸的水解速率在H35D细胞中比在H35细胞中高4至5倍。分离的溶酶体中的GH活性部分依赖于还原剂如巯基乙醇的存在。用Triton X-100使两种细胞类型的溶酶体制剂通透化会使GH活性增强10倍。H35D细胞中GH活性增强的结果是抗叶酸多聚谷氨酸浓度显著降低，在所有测试条件下，母体抗叶酸是主要的细胞内物质。与抗叶酸不同，在标准培养条件下，直至10微摩尔叶酸，两种细胞中的总细胞内叶酸浓度几乎相同。然而，在GH增加的H35D细胞中，叶酸多聚谷氨酸的链长主要由三谷氨酸和四谷氨酸组成，而在亲代细胞中则由五谷氨酸和六谷氨酸组成。在50和100微摩尔叶酸时，H35D细胞中的叶酸积累量不到H35细胞的一半，并且H35D细胞中主要的多聚谷氨酸种类是二谷氨酸至四谷氨酸。结果表明，两种H35细胞系具有相等的叶酸多聚谷氨酸合成酶，但其中一种具有增强的溶酶体GH活性，其叶酸多聚谷氨酸和抗叶酸多聚谷氨酸的量和γ-谷氨酰链长显著降低。
