Figure 4. Identification of the enzyme-butylimine intermediate in the DHSR1 and DHSRC mixtures by reduction with NaBH₃CN.

(A) fluorogram and (B) Coomassie blue staining. The DHSR1 mixture contained 10 μg enzyme, 20 μCi (0.56 nmol) of [1,8-³H]spermidine plus 1.44 nmol of unlabeled spermidine, 100 μg BSA and 100 μM NAD in 100 μl of 0.1M Gly-NaOH buffer (pH 9.0). DHSRC mixture contained 20 μg of eIF5A precursor in addition to all the components of DHSR1. After 3, 10, 20, and 30 min at 37 °C, 20 μl of the reaction mixture was removed and treated with 1 μl of NaBN₃CN (0.1 M) three times at 5 min intervals on ice. Additional carrier BSA (100 μg) was added and the mixtures were precipitated with 10 % TCA containing nonlabeled putrescine, spermidine and spermine (1 mM each). The TCA precipitates were washed two times and dissolved in 50 μl of SDS sample buffer. After SDS-PAGE, staining and destaining, the gel was treated with Amplify for 30 min, dried and exposed to Xray film at −80 °C. The experiment was repeated with similar results and a representative experiment is shown.