Wang Y, Nimec Z, Ryan T J, Dias J A, Galivan J
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Biochim Biophys Acta. 1993 Aug 7;1164(3):227-35. doi: 10.1016/0167-4838(93)90253-n.
gamma-Glutamyl hydrolase has been partially purified and characterized from conditioned culture medium of H35 hepatoma cells. Evidence for heterogeneity of the enzyme is derived from its elution as three distinct peaks of enzymatic activity when the enzyme is purified by TSK-butyl-Sepharose column chromatography. These three enzyme fractions appear to have identical catalytic properties but, as yet, the basis for their resolution is not understood. A rapid, sensitive and simple assay based on reverse-phase HPLC fluorescent detection with pre-column derivatization using o-phthalaldehyde (OPA) was developed to separate OPA-derivatives of poly-gamma-glutamates and glutamic acid. Using this assay and the standard HPLC assay for pteroylpolyglutamates, the enzyme appears to be an endopeptidase with respect to pteroylpenta-gamma-glutamate (PteGlu5), methotrexate penta-gamma-glutamate (4-NH2-10-CH3PteGlu5) and p-aminobenzoyl-penta-gamma-glutamate (pABAGlu5). The initial products are PteGlu1 (or 4-NH2-10-CH3PteGlu1 or pABAGlu1) and intact tetra-gamma-glutamate, which is subsequently degraded to glutamic acid. When penta-gamma-glutamate is the substrate, the cleavage of the gamma-bonds by the enzyme is less ordered, with the early appearance of mono-, di-, tri- and tetraglutamate. Poly-alpha-glutamate is not a substrate nor are pABA-gamma-Glu5 or penta-gamma-glutamate covalently linked to albumin. 4-NH2-10-CH3PteGlu2 or Glu5 bound to dihydrofolate reductase is not a substrate for the enzyme, offering further evidence that protein-associated poly-gamma-glutamates are poor substrates for gamma-glutamyl hydrolase from H35 hepatoma cells.
γ-谷氨酰水解酶已从H35肝癌细胞的条件培养基中部分纯化并进行了特性鉴定。该酶异质性的证据源于，当通过TSK-丁基琼脂糖柱色谱法纯化该酶时，其以三个不同的酶活性峰洗脱。这三个酶组分似乎具有相同的催化特性，但目前尚不清楚它们分离的基础。开发了一种基于反相高效液相色谱荧光检测的快速、灵敏且简单的测定方法，该方法使用邻苯二甲醛（OPA）进行柱前衍生化，以分离聚γ-谷氨酸和谷氨酸的OPA衍生物。使用该测定方法和用于蝶酰多谷氨酸的标准高效液相色谱测定方法，该酶对于蝶酰五γ-谷氨酸（PteGlu5）、甲氨蝶呤五γ-谷氨酸（4-NH2-10-CH3PteGlu5）和对氨基苯甲酰五γ-谷氨酸（pABAGlu5）似乎是一种内肽酶。初始产物是PteGlu1（或4-NH2-10-CH3PteGlu1或pABAGlu1）和完整的四γ-谷氨酸，随后其被降解为谷氨酸。当五γ-谷氨酸作为底物时，该酶对γ键的切割不太有序，单谷氨酸、二谷氨酸、三谷氨酸和四谷氨酸会较早出现。聚α-谷氨酸不是底物，与白蛋白共价连接的对氨基苯甲酸-γ-Glu5或五γ-谷氨酸也不是底物。与二氢叶酸还原酶结合的4-NH2-10-CH3PteGlu2或Glu5不是该酶的底物，这进一步证明与蛋白质相关的聚γ-谷氨酸是H35肝癌细胞γ-谷氨酰水解酶的不良底物。
