Recombinant reverse transcriptase from Rous sarcoma virus. Kinetics and inhibition of DNA polymerase activity

Preparations of Rous sarcoma virus reverse transcriptase isolated from a culture of E. coli HB101 (pMF14) and purified to homogeneity were used to study the steady state kinetics of DNA polymerization and inhibition of DNA-polymerase activity. DNA synthesis was examined using a system of poly(rA) as...

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Bibliographic Details
Published inBiokhimiia (Moscow, Russia) Vol. 60; no. 6; p. 874
Main Authors Chernov, A P, Ivanov, V A
Format Journal Article
LanguageRussian
Published Russia (Federation) 01.06.1995
Subjects
Avian Sarcoma Viruses - enzymology
Catalysis
DNA, Recombinant - biosynthesis
DNA-Directed DNA Polymerase - metabolism
Escherichia coli - genetics
Kinetics
Nucleic Acid Synthesis Inhibitors
Recombinant Proteins - antagonists & inhibitors
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Reverse Transcriptase Inhibitors
RNA-Directed DNA Polymerase - genetics
RNA-Directed DNA Polymerase - metabolism
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Summary:Preparations of Rous sarcoma virus reverse transcriptase isolated from a culture of E. coli HB101 (pMF14) and purified to homogeneity were used to study the steady state kinetics of DNA polymerization and inhibition of DNA-polymerase activity. DNA synthesis was examined using a system of poly(rA) as template, oligo(dT) as primer and dTTP as nucleotide substrate. Kinetic constants for steady state conditions were determined. The substrate initial velocity patterns point to an ordered mechanism which results in the formation of a ternary complex, in which the template-primer is the first to bind to the enzyme. Inhibition of the DNA-polymerase activity of the enzyme by various inhibitors was studied. Analysis of final products of the DNA-polymerase reaction revealed the presence of distribution syntheses of the DNA chain by the alpha alpha-subunit form of the enzyme.
ISSN:0320-9725