Biochemical and immunological properties of the DNA polymerase and RNAase H activities of purified feline leukemia virus reverse transcriptase.
Feline leukemia virus DNA polymerase was purified by ion-exchange and nucleic acid affinity chromatographies. The enzyme consists of a single polypeptide chain of mol. wt. approx. 72,000 as determined by both glycerol density gradient centrifugation and SDS-polyacrylamide gel electrophoresis. The preferred divalent cation for DNA synthesis is Mn2+ on a variety of template-primers, and its optimum concentration appears to be significantly lower than reported results of other mammalian type-C viral enzymes. The purified enzyme also contained RNAase H activity. Both DNA polymerase and RNAase H activities appear to reside on the same molecule as demonstrated by the copurification of both activities through various purification steps. The divalent cation requirement for maximum activity of RNAase H is also similar to that of the DNA polymerase. RNAase H without detectable polymerase activity was generated by a limited chymotrypsin digestion of the purified reverse transcriptase. This RNAase H activity was inhibited equally effectively as RNAase H in the intact reverse transcriptase by antisera prepared against reverse transcriptase of feline leukemia virus. These results indicate that the RNAase H catalytic activity of reverse transcriptase is distinct from the polymerase portion of the molecule. Since the RNAase H activity appears to be more stable, the measurement of RNAase H activity with a proper antibody might be useful for assaying tumor cells for the presence of the viral enzyme.
D000818:Animals; D002415:Cats; D004259:DNA-Directed DNA Polymerase / Q000032:analysis* / Q000276:immunology; D004720:Endonucleases / Q000032:analysis* / Q000276:immunology; D005256:Leukemia Virus, Feline / Q000201:enzymology*; D008970:Molecular Weight; D009500:Neutralization Tests; D012194:RNA-Directed DNA Polymerase / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D016914:Ribonuclease H; D012260:Ribonucleases / Q000032:analysis* / Q000276:immunology
D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases; D012260:Ribonucleases; D016914:Ribonuclease H