A rapid diethylaminoethyl paper disk assay for transfer RNA sulfurtransferase.
A rapid assay for tRNA sulfurtransferase from Escherichia coli was developed, reducing the time needed to determine enzyme activity from 11 to 2 h. The reaction measured is the transfer of sulfur from [35S]cysteine to acceptor sites in a thionucleotide-deficient tRNA substrate. Processing is done by binding the product, [35S]-tRNA, to DEAE-cellulose filter disks. The disks are then treated to remove unreacted [35S]cysteine, cysteine-protein adducts and [35S]cysteinyl-tRNA. The DE81 disk assay and the 11-h standard assay are shown to give identical values over a wide range of incubation times and enzyme levels. Incorporation was greater when thionucleotide-deficient tRNA was used as substrate, as compared to fully modified tRNA. [35S]-tRNA was found to be the major reaction product, although some [35S]cysteine was also bound to the filters. The major thionucleoside labeled in nucleoside digests was 4-thiouridine, as determined by Bio-Gel P2 chromatography. We also observed other labeled peaks by this method, in amounts too small for positive identification. This rapid assay should be useful in the purification and study of this uncharacterized class of tRNA modification enzymes.
Harris
C L
CL
Department of Biochemistry, West Virginia University, School of Medicine, Morgantown 26506.
Kolanko
C J
CJ
D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D010209:Paper; D012343:RNA, Transfer / Q000302:isolation & purification; D011865:Radioisotope Dilution Technique; D013462:Sulfur Radioisotopes; D013466:Sulfurtransferases / Q000378:metabolism*
D013462:Sulfur Radioisotopes; D012343:RNA, Transfer; D013466:Sulfurtransferases; C023200:tRNA sulfurtransferase