Bovine leukemia virus: purification and characterization of the aspartic protease.
To develop efficient bovine leukemia virus (BLV) protease (PR) inhibitors, pure enzyme is required. For this, we have developed a two-step chromatographic nondenaturing purification protocol of PR from virions. As expected, the purified protein presents a molecular weight (14 kDa) and a NH2 terminal end fitting with previously reported data. The enzymatic activity of BLV PR was characterized using a synthetic peptide containing a potential cleavage site of the BLV gag-pro polypeptide precursor as substrate. The protease was most active at pH 6, 40 degrees, and high salt concentration (1-2 M NaCl or ammonium sulfate). In contrast, using a natural substrate such as a human T-cell leukemia virus recombinant gag precursor, BLV PR activity was higher at a low salt concentration (0.5 M NaCl). Besides, the use of different potentially cleavable molecules revealed that PR activity may be influenced by the substrate conformational structure around the cleavage site. Replacement of the two amino acids of a synthetic substrate cleavable site by a statin residue completely inhibited the enzymatic activity of the BLV PR.
Unité INSERM 328, Université de Bordeaux II, Structures et Fonctions des Rétrovirus Humains, fondation Bergonié, France.
A
A
Menard
R Z
RZ
Mamoun
S
S
Geoffre
M
M
Castroviejo
S
S
Raymond
G
G
Precigoux
M
M
Hospital
B
B
Guillemain
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D005333:Fetus; D015683:Gene Products, gag / Q000378:metabolism; D015750:Genes, gag; D015368:Human T-lymphotropic virus 1 / Q000235:genetics; D007668:Kidney; D007700:Kinetics; D001909:Leukemia Virus, Bovine / Q000201:enzymology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009994:Osmolar Concentration; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D015183:Restriction Mapping; D012756:Sheep; D013379:Substrate Specificity; D013816:Thermodynamics
D015683:Gene Products, gag; D010455:Peptides; D016282:Aspartic Acid Endopeptidases