Possible regulation by nucleosidediphosphate kinase involvement of the synthesis of tRNA 3'-terminal -pCpCpA in mammalian cells.
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)
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Facilities for Comparative Medicine & Animal Experimentation, Niigata University School of Medicine.
D000818:Animals; D001483:Base Sequence; D002286:Carcinoma, Ehrlich Tumor; D003600:Cytosol / Q000032:analysis; D008297:Male; D051379:Mice; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D012343:RNA, Transfer / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured / Q000032:analysis
D012343:RNA, Transfer; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase; D009713:Nucleotidyltransferases