When the cytosol of
Ehrlich ascites tumor cells was fractionated by chromatofocusing in the pH range of 9 to 6, two active peaks (I and II) of
tRNA nucleotidyltransferase were obtained. Fraction I was a multiple complex with a high molecular weight (M.W. greater than 300K) and fraction II comprised components derived from fraction I. Fraction II was separated into
tRNA nucleotidyltransferase (M.W., ca. 46,000) and nucleosidediphosphate
kinase (M.W., ca. 74,000) by subsequent
Sephacryl S-200 chromatography. The two
enzymes appeared to be associated loosely with each other. Using the above fraction II or a mixture of the purified
tRNA nucleotidyltransferase and nucleosidediphosphate
kinase, it was possible to effectively synthesize the 3'-terminal -pCpCpA of
tRNA in a reaction mixture containing [3H]-
CDP plus
XTP or [3H]
ADP plus
XTP as substrate. Among the XTPs investigated,
dTTP was most effective. In addition, it was found that [3H]
AMP +
XTP also serves as a substrate. [14C]
CMP plus
XTP, however, was not utilized. From the antagonism of cold
CDP against [3H]
CTP, and that of cold
ADP and
AMP against [3H]
ATP with the purified
tRNA nucleotidyltransferase, the affinity of
CDP to the
enzyme was estimated to be 1/100 of that of
CTP, while the affinities of
ADP and
AMP to the
enzyme were 3 and 30 times higher, respectively, than that of
ATP, suggesting that the subsite which binds
ATP also binds
ADP or
AMP. The
tRNA nucleotidyltransferase, which had bound
ADP or
AMP, could not completely synthesize the 3'-terminus of
tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)