Fibroinase activity of silk gland in larval and early pupal development of the silkworm, Bombyx mori assayed with a fluorescent quenched peptide substrate

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  • Fibroinase Activity of Silk Gland in Larval and Early Pupal Development of the Silkworm, <i>Bombyx mori</i> Assayed with a Fluorescent Quenched Peptide Substrate

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Fibroinase activity of silk gland of B. mori was determined during whole larval development and in early pupa. For this purpose we synthesized highly sensitive peptide substrates, fluorescent quenched peptides, 7-methoxycoumarin-4-acetic acid (MCA)-Ala-Gly-Tyr-Gly-Ala-Gly-Lys (2,4-dinitrophenyl group (Dnp))-Gly (hereafter referred to as MCA-Tyr-Dnp) and MCA-Ala-Gly-Phe-Gly-Ala-Gly-Lys (Dnp)-Gly (MCA-Phe-Dnp) based on the cleavage specificity of fibroinase of silk gland to preferentially split the peptide bond between Gly and Ala in the fibroin peptide (Watanabe et al., 2004a) and on the tetrapeptide produced by hydrolysis of B. mori liquid fibroin by fibroinase of silk gland for 12h as a building block of substrate, which was identified in this study. The kinetic parameters of these two substrates were determined using fibroinase of silk gland from different developmental periods, i.e., from the fourth molt period and from day 1 pupa. MCA-Tyr-Dnp gave similar kcat/Km values, while MCA-Phe-Dnp gave different values. To obtain comparable data on the fibroinase activity of silk gland during B. mori development was the objective of this study, MCA-Tyr-Dnp was adopted as a substrate. Assay was conducted both at pH4.0 and at pH5.0. Similar activity levels were obtained at both pH’s. Fibroinase activity of silk gland increased directly proportional to a log scale from day 1, the first instar larva until the middle period of the fifth instar larva by 10,000-fold. Considerable activity was observed in the feeding period in each larval instar including the fifth larval instar, the latter result of which confirmed the previous results using liquid fibroin as a substrate. Activity was also observed at each molt period and a peak of activity was observed clearly at the second, third, and fourth molt periods. These observations in the earlier period of larval development of B. mori were interpreted as a further support of a dual role of fibroinase of B. mori silk gland depending on the developmental period, i.e., at the feeding period for digestion of obsolete protein synthesis machinery and cellular organells in lysosomes, and at the molt period for digestion of fibroin and sericin accumulated in the lumen contents of silk gland, respectively.<br>

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