Involvement of cathepsin B- and L-like proteinases in silk gland histolysis during metamorphosis of Bombyx mori

To identify proteinases involved in programmed cell death of the silk gland s of Bombyx mori, we measured enzyme activities in silk gland homogenates. Several peptidyl-4-methylcoumaryl-7-amides (MCAs) and bovine hemoglobin wer e used as substrates in the presence and absence of proteinase inhibitors. The hydrolysis of t-butyloxycarbonyl-Phe-Ser-Arg-MCA (Boc-FSR-MCA), benzylo xy-carbonyl-Phe-Arg-MCA (Z-FR-MCA), and Z-Arg-Arg-MCA (Z-RR-MCA) was optima l at pH 5.5, 5.0, and 5.5, respectively. It was stimulated by the sulfhydry l compounds or EDTA and inhibited by both cysteine proteinase inhibitors an d a cathepsin B-specific inhibitor, L-3-trans-(propyl-carbamoyl)oxirane-2-c arbonyl)-L-isoleucyl-L-prolin (CA-074). The hemoglobin hydrolysis at the op timum pH 3.5 was inactivated by cysteine proteinase inhibitors, but stimula ted slightly by pepstatin. The cleavage of Arg-MCA (R-MCA) and Leu-MCA (L-M CA) at optimum pH of 7.0 was strongly inhibited by an aminopeptidase inhibi tor, puromycin, and by sulfhydryl compounds. The Boc-FSR-MCA, Z-FR-MCA, Z-R R-MCA, and hemoglobin hydrolyzing activities increased in the silk glands d ramatically after cocoon formation, while the R-MCA and L-MCA cleaving acti vities declined. The results strongly suggest the involvement of cathepsin B- and cathepsin L-like proteinases in the histolysis of the silk gland dur ing metamorphosis. (C) 2001 Academic Press.