Partial purification and properties of the reticulocyte guanylating enzyme.

The guanylating enzyme which catalyzes the insertion of a guanine residue into one of the isoacceping tRNAHis of rabbit reticulocytes has been purified approximately one-hundred fold. It is free of nuclease activity. The enzyme does not catalyze the replacement of inserted radioactive guanine by unlabeled guanine, indicating that the reaction is irreversible. We have separated the histidyl-tRNA of reticulocytes into three isoacceptors. Previous work showed that the last histidyl-tRNA to elute from RPC-5 columns was the product of the guanylation reaction. This reports shows that the same late-eluting peak also contains the substrate for the guanylating enzyme, indicating that the guanine insertion reaction is chromatographically silent. The isoaccepting tRNAHis that is the substrate for the guanylating enzyme does not contain the hypermodified base known as Q. It is the other major reticulocyte tRNAHis that coantains Q, showing that at least in the reticulocyte the role of the guanylating enzyme is not the conversion of the Q form of tRNA to the homogeneic G form. The purified enzyme does not insert any base other than guanine into tRNA.