Physico-chemical and immunological properties and partial amino acid sequencing of a new metalloprotease: endoprotease Thr-N.
Previous studies have described the isolation of a new metalloprotease with a strict specificity for the amide bonds of peptide substrates having a threonine residue at the P1' position [Biochem. Biophys. Res. Commun. 256 (1999) 307]. The present work reports the physico-chemical properties of the enzyme which enable the optimal conditions for the digestion of proteins by the protease to be determined. At pH 8.2 and up to 37 degrees C, the enzyme possesses a good proteolytic activity and is stable for at least 12 h. The protease is sensitive to detergents and dithiol-reducing agents so that these chemicals must be eliminated after treatment of the protein substrate when this needs to be denatured and reduced before its hydrolysis by the enzyme. An increase in the enzymatic activity is observed in the presence of urea up to a 2.0 M concentration, beyond which the activity decreases. The enzyme can also be used in the presence of organic solvents such as acetonitrile, isopropanol or dioxane (10%, v/v) without loss of activity. Studies performed with antibodies raised against the purified endoprotease Thr-N indicated the absence of cross-immunoinactivation and cross-immunoprecipitation with all tested proteases. Also, no homology of sequence was found with the proteases indexed in the databases. Thus, our results show that endoprotease Thr-N not only represents an original protease by its unique specificity but also by its immunological and molecular properties.
Niamké
Sébastien
S
Laboratoire de Biochimie, Unité de Recherche sur la Biocatalyse, CNRS 2230, Faculté des Sciences et des Techniques, 2 rue de la Houssinière, BP 92208, F 44322 Nantes Cedex 3, France.
Guionie
Olivier
O
Guével-David
Laetitia
L
Moallic
Claire
C
Dabonne
Soumaila
S
Sine
Jean-Pierre
JP
Colas
Bernard
B
D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D020539:Sequence Analysis, Protein; D012908:Snails / Q000737:chemistry / Q000201:enzymology; D013696:Temperature
D010450:Endopeptidases; D045726:Metalloproteases; C478782:endoprotease Thr-N