Document 0106
 DOCN  M9460106
 TI    Human immunodeficiency virus type 1 (HIV-1) recombinant reverse
       transcriptase. Asymmetry in p66 subunits of the p66/p66 homodimer.
 DT    9408
 AU    Sharma SK; Fan N; Evans DB; Upjohn Laboratories, Kalamazoo, MI 49002.
 SO    FEBS Lett. 1994 Apr 25;343(2):125-30. Unique Identifier : AIDSLINE
       MED/94222184
 AB    A recombinant p66 form of human immunodeficiency virus type 1 (HIV-1)
       reverse transcriptase (RT) can be obtained [(1991) Biotechnol. Appl.
       Biochem. 14, 69-81] from crude Escherichia coli extracts by immobilized
       metal affinity chromatography (IMAC). We have analyzed the p66 HIV-1 RT,
       isolated in the presence of 0.3 M imidazole, by gel permeation HPLC on
       Superose 12. The results show that it contains two major distinct p66
       forms (24.1 min and 28.3 min peaks) which are distinguishable from the
       purified homodimeric (p66/p66) HIV-1 RT (22.2 min peak). Protein peak 1
       (24.1 min) is converted to a 22.3 min peak upon storage for 20 h at 4
       degrees C. Under identical conditions, the isolated peak 2 (28.3 min)
       appeared as a conformationally heterogeneous mixture elaborated by peaks
       at 22.3 min and 25.9 min. The protein species thus obtained were active
       in the RNA-dependent DNA polymerase and RNase H activity assays and
       produced heterodimeric HIV-1 RT upon incubation with the HIV-1 protease.
       When the IMAC-purified, imidazole-free homodimeric (p66/p66) form of the
       enzyme was incubated with 0.3 M imidazole for 16 h at 4 degrees C,
       protein peaks at 28.3 min (peak A) and 30.5 min (peak B) were isolated
       by gel permeation HPLC. While both of these p66-containing species were
       stable and displayed identical RNA-dependent DNA polymerase activities,
       the protein in peak B was only 50% active in RNase H function compared
       with the protein from peak A. These imidazole-mediated dissociation
       studies support the hypothesis of partial unfolding of one of the RNase
       H domains of the p66/p66 homodimer, suggesting that the p66 subunits are
       asymmetric in the native enzyme.
 DE    Chromatography, High Pressure Liquid  Electrophoresis, Polyacrylamide
       Gel  Escherichia coli  HIV-1/*ENZYMOLOGY  Protein Conformation  Protein
       Folding  Recombinant Proteins/CHEMISTRY/ISOLATION & PURIF/METABOLISM
       Reverse Transcriptase/*CHEMISTRY/ISOLATION & PURIF/METABOLISM
       Ribonuclease H, Calf Thymus/METABOLISM  Support, U.S. Gov't, P.H.S.
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).