Cell surface gamma-glutamyl transpeptidase in live cultures.
Cell surface gamma-glutamyl transpeptidase in live cultures.
复制标题
活培养物中的细胞表面γ-谷氨酰转肽酶。
DOI：
10.1016/0003-2697(91)90202-5
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发表时间：
1991
影响因子：
2.9
通讯作者：
DeBault,L
中科院分区：
文献类型：
--
作者：
Morgenstern,K;Hanson-Painton,O;DeBault,L
描述了用于测量粘附的活培养物中的表面可及γ-谷氨酰转肽酶活性的生理测定法。细胞表面转肽酶活性保持线性，在整个60分钟的时间过程中，在很宽的细胞密度。此外，试验条件对细胞生长潜力、细胞形态或细胞表面转肽酶活性水平均无急性或长期影响。因此，可以在增殖期间在相同的培养物中连续评估细胞表面转肽酶活性。该测定似乎是特定的细胞表面转肽酶，并可用于研究基板之间的分区和基板中的访问池的酶。该方法使用自动微量滴定板读数器对从与显色底物1-γ-谷氨酰-对硝基苯胺孵育的培养物中取出的小等份试样进行分光光度定量。微量滴定板自动读数器的使用和对细胞的最小处理允许大量培养物被测定，同时显著减少测量表面转肽酶活性所需的时间。所述测定是用于研究活培养物微环境中细胞表面可及γ-谷氨酰转肽酶催化活性的非破坏性方法。
A physiological assay for measuring surface accessible γ-glutamyl transpeptidase activity in adherent, living cultures is described. Cell surface transpeptidase activity remained linear throughout a 60-min time course over a wide range of cell densities. In addition, the assay conditions have neither acute nor long-term effects on cell growth potential, cellular morphology, or cell surface transpeptidase activity levels. As a result, cell surface transpeptidase activity can be continually evaluated in the same cultures during proliferation. The assay appears to be specific for cell surface transpeptidase and can be used to study the partitioning of the enzyme between substrate-accessible and substrate-in-accessible pools. This method utilizes an automated microtiter plate reader for the spectrophotometric quantification of small aliquots removed from cultures incubated with the chromogenic substrate l-γ-glutamyl-p-nitroanilide. The use of a microtiter plate autoreader and the minimal handling of the cells permit a large number of cultures to be assayed with a substantial reduction in the time required to measure surface transpeptidase activity. The assay described is a nondestructive means for studying cell surface-accessible γ-glutamyl transpeptidase catalytic activity within the microenvironment of the living culture.