Isolation and characterization of a single-stranded specific endoribonuclease from Ehrlich cell nucleoli.
An endoribonuclease which cleaves only single-stranded RNA has been purified from nucleoli of Ehrlich ascites tumor cells. The molecular weight of the ribonuclease is 50,000 to 52,000 as estimated from sedimentation in glycerol density gradients and by gel filtration on Sephadex G-100. The endoribonuclease requires Mg2+ or Mn2+ (0.2 mM) for optimum activity. Monovalent cations including K+, Na+, and NH+4 are inhibitory. The ribonuclease gave an apparent Km for single-stranded RNA of 30 microM. Using ribohomopolymers, we found that the enzyme could digest single-stranded, poly(C), poly(U), and poly(A) equally well, but would not degrade duplex poly(C) . poly(I) or poly(A) . poly(U). The lack of base specificity was further demonstrated using RNA sequence analysis of partial digest products of yeast 5.8 S RNA. The ribonuclease activity is sensitive to EDTA and N-ethylmaleimide, but is not inhibited by human placental RNase inhibitor. The enzyme makes endonucleolytic cleavages which generate 5'-phosphate-terminated oligonucleotides.
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002413:Cations, Divalent; D002466:Cell Nucleolus / Q000201:enzymology*; D004722:Endoribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D013379:Substrate Specificity
D002413:Cations, Divalent; D004722:Endoribonucleases; C036544:endoribonuclease, single-stranded specific