title: Cloning and sequence of a cDNA for a highly basic protease from the digestive juice of the silkworm, Bombyx mori.
abstract: A serine protease of the silkworm, Bombyx mori, with an isoelectric point of pH 10-11 and a pH optimum for succinyl-Leu-Leu-Val-Tyr-MCA degrading activity of about 10, was found in a 0.33 M NaCl-eluted fraction obtained from cation-exchange chromatography of digestive juice. The activity of the enzyme was strongly inhibited by chymostatin and PMSF, indicating that the protease is a chymotrypsin-like serine protease. The N-terminal amino acid sequence of the protease was determined, and a full-length cDNA clone (0.92 kbp) which was isolated from a midgut cDNA library was sequenced. The cDNA encodes a pre-proenzyme of 284 amino acids with a pro-segment of 50 amino acids and mature protein of 234 amino acids. From its primary structure, the predicted molecular mass of the mature protein is 24.5 kDa. A sequence comparison of the Bombyx highly basic protease with other serine proteases revealed that this enzyme is a mammalian-type serine protease with a catalytic triad consisting of His45, Asp92 and Ser186. A large number of Arg residues are encoded by the cDNA which may be responsible for its stability and/or function in the alkaline condition, by remaining charged at high pH.
authors.affiliation: Department of Applied Biology, Kyoto Institute of Technology, Japan. kotani@ipc.kit.ac.jp
authors.forename: E
authors.initials: E
authors.lastname: Kotani
authors.forename: T
authors.initials: T
authors.lastname: Niwa
authors.forename: M
authors.initials: M
authors.lastname: Tokizane
authors.forename: K
authors.initials: K
authors.lastname: Suga
authors.forename: Y
authors.initials: Y
authors.lastname: Sugimura
authors.forename: K
authors.initials: K
authors.lastname: Oda
authors.forename: H
authors.initials: H
authors.lastname: Mori
authors.forename: T
authors.initials: T
authors.lastname: Furusawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D002417:Cattle; D005591:Chemical Fractionation; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004064:Digestive System / Q000201:enzymology; D008969:Molecular Sequence Data; D012685:Sepharose / Q000031:analogs & derivatives; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D012685:Sepharose; D012697:Serine Endopeptidases