title: Purification and enzymological characterization of murine neurotrypsin.
abstract: An increasing number of studies indicate that serine proteases play an important role in structural plasticity associated with learning and memory formation. Neurotrypsin is a multidomain serine protease located at the presynaptic terminal of neurons. It is thought to be crucial for cognitive brain functions. A deletion in the neurotrypsin gene causes severe mental retardation in humans. For a biochemical characterization, we produced murine neurotrypsin recombinantly in a eukaryotic expression system using myeloma cells. From the culture medium we purified neurotrypsin using heparin-, hydrophobic interaction- and immobilized metal affinity chromatography. For an enzymological characterization two fragments of agrin containing the natural cleavages sites of neurotrypsin were used as substrates. The highest catalytic activity of neurotrypsin was observed in the pH range between 7.0 and 8.5. Calcium ions were required for neurotrypsin activity and an ionic strength exceeding 500 mM decreased substrate cleavage. Site-specific mutations of the amino acids flanking the scissile bonds showed that cleavage is highly specific and requires a basic amino acid preceded by a glutamate residue on the N-terminal side of the scissile bond. This sequence requirement argues for a unique substrate binding pocket of neurotrypsin. This observation was further substantiated by the fact that almost all tested serine protease inhibitors except dichloroisocoumarin and PMSF did not affect neurotrypsin activity.
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland.
authors.forename: Raymond
authors.initials: R
authors.lastname: Reif
authors.forename: Susanne
authors.initials: S
authors.lastname: Sales
authors.forename: Birgit
authors.initials: B
authors.lastname: Dreier
authors.forename: Daniel
authors.initials: D
authors.lastname: Lüscher
authors.forename: Jens
authors.initials: J
authors.lastname: Wölfel
authors.forename: Claudio
authors.initials: C
authors.lastname: Gisler
authors.forename: Antonio
authors.initials: A
authors.lastname: Baici
authors.forename: Beat
authors.initials: B
authors.lastname: Kunz
authors.forename: Peter
authors.initials: P
authors.lastname: Sonderegger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C108493:neurotrypsin