HTLV-I protease cleavage of P19/24 substrates is not dependent on NaCl concentration.
Understanding the factors that affect the activity of Human T-cell Leukemia Virus type I (HTLV-I) protease is essential for the discovery of inhibitors to be used for the treatment of HTLV-I infection, but little has been reported on the protease to date. Here we report the production of HTLV-I protease in purified yields greater than 150 mg/L, determination of its extinction coefficient, and determination of the optimum conditions for cleavage of the p19/24 substrates (DABCYL)-(GABA)-PQVL-Nph-VMH-(EDANS), (DABSYL)-(GABA)-PQVL-Nph-VMH-(EDANS), and (DABSYL)-(GABA)-PQVLPVMH-(EDANS). The highest activity was found at pH 5.2-5.3 and 37 degrees C. There was no effect on activity upon change in sodium chloride concentration from 0 to 1500 mM. The values of K(m) and k(cat) for cleavage of these substrates by the protease with and without the histidine tag were determined.
Ha
Julie J
JJ
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA.
Gaul
David A
DA
Mariani
Victoria L
VL
Ding
Y Shirley
YS
Ikeda
Richard A
RA
Shuker
Suzanne Beckham
SB
D016282:Aspartic Acid Endopeptidases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D005456:Fluorescent Dyes; D009842:Oligopeptides; D012965:Sodium Chloride; D016282:Aspartic Acid Endopeptidases; C079711:HTLV-1 protease