Serological analysis and characterization of calf thymus ribonuclease H IIb.
Ribonuclease H IIb, which seems to play a physiological role during transcription, was purified from calf thymus tissue. A polyclonal antibody, raised against the most purified ribonuclease H IIb fraction, recognizes in crude extracts almost exclusively a 52-kDa protein band. By immunoaffinity chromatography and immunoprecipitation experiments, we are able to deplete enzyme extracts from the crossreacting 52-kDa protein band and from ribonuclease H IIb activity. Enzyme activity is eluted from the immunoaffinity matrix in association with a 52-kDa protein under denaturing conditions. Immunoaffinity chromatography enables us also to calculate a purification factor of around 20,000 from the crude extract. The native molecular mass for the enzyme of around 45 kDa, as determined by gel filtration, suggests that calf thymus ribonuclease H IIb is most probably monomeric. The enzyme possesses an isoelectric point of 7.0. It requires Mg2+ ions for activity, is inhibited by N-ethylmaleimide, and exhibits a pH optimum of 9.0-9.5. The enzyme releases oligoribonucleotides with 3'-OH and 5'-phosphate ends, probably in an exonucleolytical manner. The third largest subunit of yeast RNA polymerase A (I) displays ribonuclease H activity [Huet et al. (1976) Nature 261, 431-433]. We discuss our findings in the light of a possible association of ribonuclease H IIb and RNA polymerase A (I) in higher eukaryotes.
Vonwirth
H
H
Lehrstuhl für Allgemeine Genetik, Universität Tübingen, Federal Republic of Germany.
Frank
P
P
Büsen
W
W
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D004722:Endoribonucleases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D016914:Ribonuclease H; D013950:Thymus Gland / Q000201:enzymology*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D004722:Endoribonucleases; D016914:Ribonuclease H