Enzymatic properties of fibroinase of silk gland from day one pupa of the silkworm, Bombyx mori

Enzymatic properties of fibroinase of silk gland from day one B. mori pupa were investigated to obtain additional comparable data with those of the fibroinases of silk gland from the fourth molt period (Watanabe et al., 2004a) and from the spinning period (Sutthikhum et al.,2004a, b). Fibroinase of silk gland from day one pupa hydrolyzed liquid fibroin efficiently. Subunit and native molecular masses were estimated as 34.2 and 19.8 kDa. Optimum pH was at pH 3.71 and at pH 5.5 with liquid fibroin and benzyloxycarbonyl (Z)-Phe-Arg-4-methyl-coumaryl-7-amide (MCA) being used as substrates. Proteinase inhibitor concentrations (nM) required to inhibit activity by 50% were: leupeptin (1.14), E-64 (1.22), Z-Phe-Phe-CHNsub(2) (3.46), antipain (8.26), chymostatin (10.1), TLCK (12.5), Z-Phe-Ala-CHNsub(2) (29.3), TPCK (577.5), iodoacetic acid (1096) and pepstatin (37077) with no inhibition by APMSF. Fibroinase stored at 30 deg C for 1 h at pH's 4.0 and 5.0 was stable retaining half the initial activity but was unstable at pH's 6.0 and above. These properties clearly indicate that fibroinase of silk gland from day one pupa is a cathepsin L-like cysteine proteinase. Properties of fibroinase of silk gland from day one pupa determined with liquid fibroin as a substrate were similar to those of the fibroinase of silk gland from the fourth molt period. However the properties determined with Z-Phe-Arg-MCA as a substrate, fibroinase of silk gland from day one pupa was similar to the fibroinase of silk gland from spinning period. These differences should result from the differences in physical properties of the substrate which affected the responses of fibroinase of silk gland from respective developmental period. Based on several evidences, fibroinase of silk gland that is similar in enzymatic properties to the fibroinase of silk gland from day one pupa is highly likely responsible for enzymatic digestion of fibroin and sericin remaining in the silk gland of B. mori pupa prior to apoptosis of pupal silk gland.