title: Evaluation of cathepsin B activity for degrading collagen IV using a surface plasmon resonance method and circular dichroism spectroscopy.
abstract: Evaluation of cathepsin B activities for degrading collagen IV and heat-denatured collagen IV (gelatin) were performed by surface plasmon resonance (SPR) and circular dichroism (CD) measurements. The optimal pH of cathepsin B activity for degrading each substrate was around 4.0. The ΔRU(15 min), which is a decrease in the SPR signal at 15 min after injection of cathepsin B, was smaller for collagen IV than for heat-denatured collagen IV owing to the presence of triple-helical conformation. An unstable nature of the triple-helical conformation of collagen IV at pH 4.0 was shown by the CD study. Degrading collagen IV by cathepsin B was facilitated owing to a local unwinding of the triple-helical conformation caused by proteolytic cleavage of the non-helical region. The concentration dependence of the initial velocity for degrading collagen IV by cathepsin B at pH 4.0 was biphasic, showing that cathepsin B at low concentration exhibits exopeptidase activity, while the enzyme at high concentration exhibits endopeptidase activity. The kinetic parameters for the exopeptidase activity of cathepsin B toward collagen IV and heat-treated collagen IV were evaluated and discussed in terms of the protease mechanism.
authors.affiliation: Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan; School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Shoji
authors.affiliation: Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan.
authors.forename: Mitsutaka
authors.initials: M
authors.lastname: Kabeya
authors.affiliation: Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan.
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Ishida
authors.affiliation: School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Yanagida
authors.affiliation: School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Shibusawa
authors.affiliation: Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan. Electronic address: sugawara@chs.nihon-u.ac.jp.
authors.forename: Masao
authors.initials: M
authors.lastname: Sugawara
mesh_terms: D002401:Cathepsin B / Q000378:metabolism*; D002942:Circular Dichroism / Q000379:methods*; D024141:Collagen Type IV / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D024141:Collagen Type IV; D002401:Cathepsin B
keywords: CD spectroscopy; Cathepsin B; Collagen IV; Proteolytic degradation; Suface plasmon resonance