Fibroinase activity in Bombyx mori silk gland in the larval-pupal development and its partial purification from spinning larva

Fibroinase activity in the B. mori silk gland was known to occur at the fourth molt period in the fourth instar larva (Sumida et al., 1993b) and at day 1 in the pupa (Sumida et al., 1993a). In order to know if fibroinase activity is present in the silk gland in other developmental periods, i.e., feeding and spinning periods, we developed two methods; a homogenization method of silk gland containing large amount of fibroin and sericin in its lumen contents and a quantitative assay method of fibroinase using liquid fibroin as a substrate. The activity was detectable in the silk gland in the feeding and spinning periods; it was low in the early fifth instar larva, rose rapidly in the middle of the instar and reached a plateau just before the onset of spinning and this level was maintained during spinning. Silk gland fibroinase was partially purified from the spinning larva. The final preparation hydrolyzed fibroin efficiently. Subunit molecular mass was 32.5 kDa and N-terminal amino acid sequence was LPEQVDWRKHGA. Similar properties were observed in the B. mori fibroinase of silk gland purified from the fourth instar larva and the day 1 pupa (unpublished data). Optimum pH was at 5.85 when the liquid fibroin was used as a substrate, the value of which was higher than those of the fibroinase from the fourth instar larva and the day 1 pupa. The concentrations of proteinase inhibitors required to inhibit fibroinase activity completely using liquid fibroin as a substrate and expressed in micro M were: E-64, 0.92; leupeptin, 1.6; TLCK, 2.2; chymostatin, 2.6; antipain, 24.0: TPCK, 94.0; pepstatin. 100.0 and iodoacetic acid, 680.0 with no inhibition by APMSF. These results indicate that fibroinase of silk gland present in the spinning B. mori larva is a cathepsin L-like cysteine proteinase.