Digestive proteolysis in Tenebrio molitor larvae takes place in the midgut, which can be divided into an anterior part (AM) with an average pH 5.6, and a posterior part (PM), average pH 7.9 (Terra et al., 1985. Insect Biochem. 15:443-449). The major proteolytic activity (64%) is located in the AM. The spectrum of proteinases in the AM and PM was studied using specific p-nitroanilide and protein substrates, inhibitors and activators of the active site, activity electrophoresis and different types of chromatographic fractionation. The major activity in the AM was due to cysteine proteinases. All five fractions of activity were inhibited by E-64 and iodoacetamide, displayed maximal activity at pH 5.0-7.0, were stable at pH 5.6 and lost up to 80% of activity at pH 7.9. The major activity in the PM was attributed to serine proteinases, which were also present in the AM. The serine proteinases of T. molitor were composed of 2 trypsin-like activities which were inhibited by TLCK, a single chymotrypsin-like activity inhibited by TPCK, and 2 fractions of serine proteinases from unidentified families (one presumably subtilisin-like), inhibited by PMSF but insensitive to ketones. The pH-optima of serine proteinases were between 7.0 and 10.0. The results of this research show that digestive proteolysis in T. molitor larvae is performed by a wide range of different proteinases. The multiplicity of cysteine proteinases in the AM apparently points to their important role in digestion, and combined with instability in the PM environment, suggests their involvement in the regulation of the initial stages of food proteolysis. Compartmentalization of digestive proteinases and their probable sequential action is determined by the sharp pH gradient along the midgut.
The work was supported by RFBR (02-04-48808) and CRDF (RB2-2396-MO-02).
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