A rapid assay for Escherichia coli 4-thiouridine-tRNA sulfurtransferase.

A simple filter paper assay for the measurement of Escherichia coli 4-thiouridine-tRNA sulfurtransferase activity is described. The assay includes the following procedures: (a) incubation of enzyme with appropriate substrates including unfractionated yeast tRNA and ³⁵S]cysteine, (b) reisolation of tRNA, and (c) binding of tRNA to ion exchange filter papers. The assay can be routinely performed with relatively small sample volumes (0.1 ml) and completed within 14 h. Proof of the validity of the assay is based in part on two experimental observations: (1) tRNA is the predominant ³⁵S-labeled species remaining bound to the filter after extensive washing, and (2) 4-thiouracyl is the predominant thiolated base formed during the assay.