Wang Y, Dias J A, Nimec Z, Rotundo R, O'Connor B M, Freisheim J, Galivan J
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
Adv Enzyme Regul. 1993;33:207-18. doi: 10.1016/0065-2571(93)90019-a.
gamma-Glutamyl hydrolase is a ubiquitous enzyme that has the capacity to cleave gamma-glutamyl bonds of cellular folyl- and antifolylpoly-gamma-glutamates. This study has revealed that the enzyme is secreted by primary cultures of rat hepatocytes and by H35 hepatoma cells. It was found that more than 99% of the total enzyme from H35 cells accumulated in the medium after 48 hr incubation with the serum-free medium. The cells were shown to remain intact during the secretion period since lactate dehydrogenase, dihydrofolate reductase and lysosomal hydrolases other than gamma-glutamyl hydrolase were retained within the cell. When PteGlu5 (folylGlu4) is used as a substrate the initial product is PteGlu (folate), and there is no appearance of intermediate chain length pteroyl polyglutamates. Therefore, the secreted and cellular gamma-glutamyl hydrolase from hepatoma cells appears to be an endopeptidase. Polyclonal antibodies to the poly-gamma-glutamate substrates of the enzyme were prepared and characterized. The antibodies recognize the structural differences between alpha- and gamma-glutamyl linkages but appear equally active with PteGlu5 and its analogs such as 4-NH2-10-CH3PteGlu5 and pABAGlu5. The affinity of the antibodies is related to the gamma-glutamyl structure since L-glutamic acid, folate or p-aminobenzoic acid are inactive with the antibodies. Furthermore, poly-gamma-glutamate has lower affinity for the antibodies than the poly-gamma-glutamate derivatives of PteGlu, 4-NH2-10-CH3PteGlu or pABA.
γ-谷氨酰水解酶是一种普遍存在的酶，能够裂解细胞内叶酰聚谷氨酸和抗叶酸聚谷氨酸的γ-谷氨酰键。本研究表明，该酶由大鼠肝细胞原代培养物和H35肝癌细胞分泌。研究发现，在无血清培养基中孵育48小时后，H35细胞中超过99%的总酶积累在培养基中。由于乳酸脱氢酶、二氢叶酸还原酶和除γ-谷氨酰水解酶外的溶酶体水解酶保留在细胞内，因此在分泌期间细胞保持完整。当以PteGlu5（叶酰谷氨酸4）为底物时，初始产物为PteGlu（叶酸），未出现中间链长的蝶酰多谷氨酸。因此，肝癌细胞分泌的和细胞内的γ-谷氨酰水解酶似乎是一种内肽酶。制备并表征了针对该酶的聚γ-谷氨酸底物的多克隆抗体。这些抗体能够识别α-和γ-谷氨酰键之间的结构差异，但对PteGlu5及其类似物如4-NH2-10-CH3PteGlu5和pABAGlu5的活性似乎相同。抗体的亲和力与γ-谷氨酰结构有关，因为L-谷氨酸、叶酸或对氨基苯甲酸与抗体无活性。此外，聚γ-谷氨酸对抗体的亲和力低于PteGlu、4-NH2-10-CH3PteGlu或pABA的聚γ-谷氨酸衍生物。
