title: Re-examination of a α-chymotrypsin-solubilized laccase in the pupal cuticle of the silkworm, Bombyx mori: Insights into the regulation system for laccase activation during the ecdysis process.
abstract: The laccase in the pupal cuticle of the silkworm, Bombyx mori, is thought to accumulate as an inactive precursor that can be activated stage-dependently. In this study we isolated an 81-kDa laccase from cuticular extract of B. mori that was prepared by digestion of the pupal cuticles with α-chymotrypsin. The mass spectrometric analysis of the purified protein indicates that this 81-kDa laccase is a product of the Bombyx laccase2 gene. The purified 81-kDa laccase (α-chymotrypsin-solubilized Bombyx laccase2: Bm-clac2) has an N-terminal sequence of RNPADS that corresponds to Arg146 to Ser151 of the deduced protein sequence of Bmlaccase2 cDNA, indicating that Bm-clac2 lacks the N-terminal part upstream from residue Arg146. Bm-clac2 shows enzymatic activity, but its specific activity is increased around 17-fold after treatment with trypsin, which involves cleavage of peptide bonds at the C-terminal region. We also found that the activity of Bm-clac2 is increased in the presence of isopropanol. In previous reports, proteolytic processing has been hypothesized as a system for laccase activation in vivo, but the present result implies that this type of processing is not the only way to convert Bm-clac2 to the high-activity enzyme.
authors.affiliation: Department of Biological Sciences, Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192-0397, Japan. Electronic address: asano-tsunaki@tmu.ac.jp.
authors.forename: Tsunaki
authors.initials: T
authors.lastname: Asano
authors.affiliation: Department of Chemistry, Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Taoka
authors.affiliation: Department of Chemistry, Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Yamauchi
authors.affiliation: Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA.
authors.forename: R
authors.initials: R
authors.lastname: Craig Everroad
authors.affiliation: Department of Biological Sciences, Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Seto
authors.affiliation: Department of Chemistry, Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Isobe
authors.affiliation: Division of Cellular Biosignal Sciences, Department of Biochemistry, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Kamo
authors.affiliation: Division of Cellular Biosignal Sciences, Department of Biochemistry, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Chosa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D042845:Laccase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018978:Molting; D011679:Pupa / Q000201:enzymology
chemical_list: D042845:Laccase; D002918:Chymotrypsin; C117608:alpha-chymotrypsin
keywords: Activation; Laccase; Phylogenetic analysis