A GFP-transfected HFWT cell line,GHINK-1, as a novel target for non-RI activated natural killer cytotoxicity assay

An anchorage-dependent Wilms' tumor cell line, HFWT, has been found to be extremely susceptible to human natural killer (NK) cells. Here we established a transfectant of HFWT with the green fluorescence protein (GFP) gene, designated GHINK-1 cells, to apply to the activated NK cytotoxicity assay without radioisotope labeling. After being co-cultured with CD3 CD56+ NK cells, GHINK-1 cells released GFP into the medium. The intensity of the fluorescence from the released GFP correlated almost exactly with the radioactivity of a standard 51Cr-release assay performed with suspension-cultured K562 cells. Because it does not require separation of the remaining live target cells by centrifugation, the non-radioisotopic GFP release assay with GHINK-1 cells is a convenient alternative for monitoring human activated NK killing activity.     

A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8

Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-¹⁴C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The K_M and k_cat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min^?1, respectively.