Figure 4.

End-point fluorescein cadaverine assay for Ca²⁺-dependent TGase activity in a liver homogenate and in liver mitochondria prepared from WT mice. The reaction mixtures, which contained Ca²⁺, fluorescein cadaverine, N,N-dimethylcasein, and DTT, were incubated for 2 h at 37 °C. The increase in fluorescence resulting from covalent attachment of fluorescein cadaverine to N,N-dimethylcasein was then determined (see the Experimental Procedures). Reaction mixtures labeled H, M, and TG contained 150 μg of homogenate protein, 150 μg of mitochondrial protein, or 2.5 μg of commercial guinea pig TGase 2 protein, respectively. Controls (blanks, H + EDTA, M + EDTA, TG + EDTA) contained the complete reaction mixture plus liver homogenate, mitochondria, or purified TGase 2, plus EDTA. The homogenate and mitochondria were prepared from fresh liver. The data are from three separate experiments. TGase 2 was used as a positive control (see the Experimental Procedures). RFU, relative fluorescence in arbitrary units; TG, commercial guinea pig TGase 2. Note the break on the y axis for RFU of TG in the absence of EDTA. The RFU values for H versus H + EDTA, for M versus M + EDTA, and for TG versus TG plus EDTA are significantly different for each pair with p values of 0.03, 0.01, and 0.001, respectively, by the paired t test.