Affinity labelling and characterization of the ppp(A2'p)nA-dependent endoribonuclease from different mammalian sources.
The ppp(A2'p)nA-dependent endoribonucleases from a number of different mammalian sources have been investigated. The enzyme from reticulocyte lysates shows optimal activity of 50-150 mM KCl and requires the presence of Mg2+. Whilst the enzyme is inactivated after passage of reticulocyte lysates through Sephadex columns in the absence of ATP, it retains full activity provided ATP is included in the column buffer. The activity of the partially purified nuclease was unaffected by the addition of reticulocyte RNase inhibitor, which, in contrast, effectively inhibited other endogenous endonucleases. The ppp(A2'p)nA-dependent Rnase co-purified with a ppp(A2'p)nA-binding protein and with a protein which could be specifically covalently labelled with an oxidised radioactive analogue of ppp(A2'p)nA. This covalent labelling could be carried out either with the partially purified RNase or in crude extracts from rabbit reticulocytes, mouse Krebs and Ehrlich ascites tumour cells and human lymphoblastoid (Daudi) or HeLa cells. In each case the affinity labelled protein migrated to a position corresponding to a apparent molecular weight of about 85 000 on electrophoresis on dodecylsulphate/polyacrylamide gels. In all cases labelling could be prevented by the addition of an excess of unlabelled ppp(A2'p)nA but not, for example, by a similar excess of the biologically inactive dimer ppp(A2'p)'A. It is concluded that the RNase and ppp(A2'p)nA binding activities are likely to reside in the same molecule.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000097:blood; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002287:Carcinoma, Krebs 2 / Q000201:enzymology*; D002460:Cell Line; D004720:Endonucleases / Q000097:blood* / Q000302:isolation & purification; D004722:Endoribonucleases; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D007945:Leukemia, Lymphoid; D051379:Mice; D009841:Oligonucleotides / Q000097:blood*; D009843:Oligoribonucleotides / Q000097:blood*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D012260:Ribonucleases / Q000097:blood* / Q000302:isolation & purification; D013045:Species Specificity
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D000255:Adenosine Triphosphate; D004720:Endonucleases; D004722:Endoribonucleases; D012260:Ribonucleases; C034663:ppp(A2'p)nA-dependent endoribonuclease protein, mouse