       Document 0451
 DOCN  M9640451
 TI    Recombinant human immunodeficiency virus type 1 reverse transcriptase is
       heterogeneous.
 DT    9604
 AU    Wilson JE; Wright LL; Martin JL; Haire SE; Ray PH; Painter GR; Furman
       PA; Division of Biochemistry, Burroughs Wellcome Co., Research; Triangle
       Park, North Carolina, USA.
 SO    J Acquir Immune Defic Syndr Hum Retrovirol. 1995 Jan 1;11(1):20-30.
       Unique Identifier : AIDSLINE MED/96130043
 AB    Recombinant wild type (wt) and T215Y HIV-1 reverse transcriptase (RT)
       were isolated using three methods designated A, B, and C. The three
       samples of wt RT were kinetically indistinguishable with respect to dTTP
       turnover on poly(rA).p(dT)10. However, whereas the kinetic constants for
       dTTP and AZTTP for both T215Y B and T215Y C were similar to those of wt
       protein, T215Y A exhibited a twofold increase in Km value for dTTP and a
       13-fold increase in Ki value for AZTTP with respect to wt protein
       purified in the same manner. We further investigated this observation by
       studying the denaturation of wt RT by urea. The urea denaturation curves
       monitored by fluorescence and circular dichroism spectroscopy were not
       coincident with the denaturation curve monitored by enzyme activity and
       yielded Cm values (the concentration of urea at which 50% of the protein
       is denatured) of 4.1 and 2.0 M urea, respectively. The noncoincidence of
       the transition curves reflects two separable, sequential, noncooperative
       conformational changes in the molecule: (a) from a catalytically active
       to an inactive conformation, and (b) from a catalytically inactive to a
       denatured, unfolded conformation. We therefore used denaturation as
       detected by changes in enzyme activity to compare the conformational
       stability of the three samples of wt and T215Y RT A, B, and C. The Cm
       values for T215Y RT did not differ from those of the respective wt;
       however, differences in Cm values were noted depending on how the
       protein was isolated. This suggested that the heterogeneity of the
       recombinant RT was due to small differences in conformation at or near
       the active site.
 DE    Antiviral Agents/PHARMACOLOGY  Chromatography, Affinity  Chromatography,
       Gel  Electrophoresis, Polyacrylamide Gel  Enzyme Stability  Escherichia
       coli/ENZYMOLOGY/GENETICS  Human  HIV-1/*ENZYMOLOGY/GENETICS  Kinetics
       Protein Conformation  Protein Denaturation/DRUG EFFECTS  Recombinant
       Fusion Proteins/CHEMISTRY/GENETICS/ISOLATION & PURIF  RNA-Directed DNA
       Polymerase/CHEMISTRY/*GENETICS/ISOLATION & PURIF  Spectrometry,
       Fluorescence  Thymine Nucleotides/PHARMACOLOGY  Urea/PHARMACOLOGY
       Zidovudine/ANALOGS & DERIVATIVES/PHARMACOLOGY  JOURNAL ARTICLE

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

