Simian immunodeficiency virus reverse transcriptase. Purification and partial characterization.
Native reverse transcriptase from simian immunodeficiency virus was purified from virus with good recovery to near homogeneity. The optimum reaction conditions of the enzyme were determined with respect to divalent cations, pH and ionic strength. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA synthesis activity. In addition, we could demonstrate an associated RNase H activity. Employing novel assay conditions, activated DNA as a heteropolymeric substrate was used more efficiently than the homopolymeric substrate poly(rA).oligo(dT) which in turn was used twofold more effectively as the template primer than poly(dC).oligo(dG). Other homopolymeric substrates, including poly(rC).oligo(dG), were also tested but were found to be poorly used by the reverse transcriptase. The Miachaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Simultaneously, using dideoxyadenosine triphosphate as nucleotide analogue, we could show that this compound acts as a competitive inhibitor with respect to dATP, whereas it acts as a non-competitive inhibitor with respect to the other nucleotides. Gel electrophoretic analysis showed the enzyme to consist of two polypeptides with apparent molecular masses of 64 and 48 kDa. Using activity gel electrophoresis, we were able to demonstrate that both subunits exhibit DNA synthesis activity.
Paul Ehrlich Institut, Langen/Frankfurt, FRG.
G
G
Kraus
E
E
Behr
M
M
Baier
H
H
König
R
R
Kurth
D000818:Animals; D002522:Chlorocebus aethiops; D004247:DNA / Q000096:biosynthesis; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D054306:Dideoxynucleotides; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification*; D015302:Simian Immunodeficiency Virus / Q000032:analysis / Q000201:enzymology* / Q000378:metabolism; D014764:Viral Proteins / Q000302:isolation & purification*
D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D009711:Nucleotides; D010455:Peptides; D014764:Viral Proteins; C037773:2',3'-dideoxyadenosine triphosphate; D004247:DNA; D012194:RNA-Directed DNA Polymerase