Purification and Characterization of Fibroinase, a Cathepsin L-Like Cysteine Proteinase, from the Silk Gland in the Fourth Instar Bombyx mori Larva at the Fourth Molt Period, Stage D₂

Abstract

In order to identify fibroinase in the silk gland of B. mori, fibroinase was purified to homogeneity from the silk gland in the fourth instar B. mori larva at the fourth molt period, stage D₂. Optimum pH was 4.7 as determined with Z-Phe-Arg-MCA which was a good substrate for fibroinase. Concentrations of proteinase inhibitors required to inhibit 50% of activity and expressed in nM were: leupeptin (0.833), E-64 (4.57), Z-Phe-Phe-CHN₂ (6.97), TLCK (10.1), antipain (11.5), chymostatin (13.7), Z-Phe-Ala-CHN₂ (43.3), TPCK (467) and iodoacetic acid (3176); no inhibition by pepstatin and APMSF. Fibroinase stored at pHs 4.0 and 5.0 was fairly stable and unstable at the neutral and alkaline pHs. Purified fibroinase hydrolyzed liquid fibroin, and primary cleavage site was a peptide bond between Gly and Ala. Cleavage sites of oxidized β-insulin chain were those cleaved by cathepsin L. Subunit molecular mass was 34.9kDa and native molecular mass 31.6kDa, and fibroinase was considered to consist of a monomer with molecular mass of approx. 35kDa. N-terminal amino acid sequence was Leu-Pro-Glu-Gln-Val-Asp-Trp-Arg-Lys-His. These results indicate that fibroinase in the silk gland from the fourth instar B. mori larva at the fourth molt period, stage D₂, is cathepsin L-like cysteine proteinase.