title: Purification and characterization of a protease degrading 30 kDa yolk proteins of the silkworm, Bombyx mori.
abstract: The second major yolk proteins, 30 kDa proteins (30kPs) of the silkworm, Bombyx mori, which have been provided during oogenesis, are kept continuously unused during embryogenesis and are utilized just before larval hatching. The crude extracts of newly hatched larvae cleaved 30kPs in an in vitro incubation system. A protease was highly purified from newly hatched larvae using ammonium sulfate precipitation, gel filtration and ionic exchange chromatography, and non-denaturing-polyacrylamide gel electrophoresis (ND-PAGE). The protease shared the NH2-terminal amino acid sequence conserved in many serine proteases, and the apparent molecular mass was estimated to be approximately 600 kDa by gel filtration column chromatography. The enzymatic activity was strongly inhibited by elastatinal and diisopropyl fluorophosphate (DFP), indicating that this protease is an elastase-like serine protease. The protease selectively hydrolysed 30kP-1 and 30kP-4 between Ser6 and Ala7, but could not attack other 30kPs such as 30kP-2, 30kP-3 and 30kP-5. Consequently, the protease characterized in the present study is a unique protease which may be specialized for the selective degradation of yolk proteins in silkworm eggs.
authors.affiliation: Laboratory of Sericulture and Entomoresouces, School of Agricultural Sciences, Nagoya University, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Maki
authors.forename: O
authors.initials: O
authors.lastname: Yamashita
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D012831:Bombyx / Q000201:enzymology*; D004527:Egg Proteins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D004527:Egg Proteins; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases