Stabilization and activation of recombinant human immunodeficiency virus-1 reverse transcriptase-P66.
Human immunodeficiency virus-1 reverse transcriptase-p66 is surprisingly unstable at 4 degrees C in a typical reverse transcriptase buffer that provides complete stability when enzyme is frozen at -70 degrees C. Incorporation of (rA)n(dT)12-18 template-primer in the buffer vastly improved solution stability of dilute enzyme. Incorporation of 1.0 M ammonium phosphate in the buffer promoted an unexpected and reproducible approximately 260% activation of enzyme. In addition, even enzyme that had been inactivated to 13% of its initial activity could be reactivated to the same approximately 260% higher activity level indicating a reversible interconversion of two forms of the enzyme. The effects of chaotropic and antichaotropic salts coupled with a prior observation of p66 monomer-dimer equilibrium provide suggestive evidence that these two forms of enzyme are monomeric and dimeric p66.
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
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Rowley
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Kenyon
D004789:Enzyme Activation; D004795:Enzyme Stability; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012492:Salts; D013698:Templates, Genetic
D011994:Recombinant Proteins; D012492:Salts; D012194:RNA-Directed DNA Polymerase