Stabilization and activation of recombinant human immunodeficiency virus-1 reverse transcriptase-P66

Biochem Biophys Res Commun. 1990 Mar 16;167(2):673-9. doi: 10.1016/0006-291x(90)92078-e.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Enzyme Activation
  • Enzyme Stability
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • Hot Temperature
  • Kinetics
  • Molecular Weight
  • Plasmids
  • RNA-Directed DNA Polymerase / genetics
  • RNA-Directed DNA Polymerase / metabolism*
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Salts
  • Templates, Genetic

Substances

  • Recombinant Proteins
  • Salts
  • RNA-Directed DNA Polymerase