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ABSTRACT
Gamma-glutamyl transpeptidase (GGT, EC 2.3.2.2) - a membrane-associated enzyme is a main regulator of glutathione (GSH) levels. The enzyme is not only the inhibitor of apoptosis induced by oxidative stress, but it is engaged in anti-tumor drug cell resistance and is considered as a marker for neoplastic changes, cell differentiation and aging. The surface and total activities of GGT in unfixed, fixed with para-formaldehyde vapors P cells (human fetal lung-derived cells) and cells lyzates were measured and compared using the γ-Glu-4-nitroanilide substrate. Our results showed that the surface activity was higher than the total, which might be due to the desegregation of the enzyme molecules upon mechanical cell homogenization and at the same time, after cell fixation in para-formaldehyde vapors it was decreased twice. Thus, methods including homogenization and/or aldehyde fixation are not suitable for the study of GGT activity.
In addition, visualization of GGT activity was tested using the fluorogenic substrate γ-Glu-4-hydrazido-N-hexyl-1,8- naphthalimide (γ-Glu-HHNI). Our results pointed out that GGT could not be visualized in fixed P (normal diploid) cells using this substrate probably as a result of the lower reactivity of the enzyme towards hydrazide substrates in comparison to amide substrates. On the other hand, GGT activity was well demonstrated with the same substrate in SK-MES-1 cells (human squamous cell carcinoma). Consequently, the novel fluorogenic substrate γ-Glu-HHNI could be used for diagnostic purposes on the basis of not-detectable/detectable GGT activity in normal and pathologically altered lung epithelial cells respectively.