Original Russian Text Originally published in Biochemistry (Moscow) On Line Papers in Press, as Manuscript BM04 310

Trypsin like proteinases play an important role in digestion in most insects studied [1 4]. They are also involved in processing of insecticide toxins of Bacillus thuringiensis, and this underlines protease dependent resistance of these species to the toxins In this paper, we describe a relatively simple method for purification of the main trypsin like enzyme of the posterior part of the midgut of yellow mealworm (Tenebrio molitor) larvae (TmT1) and characterize the proteinase of this stored product pest. MATERIALS AND METHODS Yellow mealworm (Tenebrio molitor) larvae were reared on a mixture of wheat flour, bran, and brewer's yeast and transferred to milled oat flakes (Raisio, Finland) 1 1.5 weeks prior to dissection. Proteinase purification. Isolated larvae midguts were separated into anterior and posterior parts. The collected posterior parts were homogenized in 0.14 M NaCl and centrifuged at 15,000g for 10 min. The supernatant dia lyzed against 20 mM K,Na phosphate buffer, pH 6.9, containing 0.02% NaN 3 , was centrifuged again and sub jected to batch chromatography on DEAE Sephadex A 50 using the same buffer. The filtrate containing the Vol. 70, No. 3, 2005, pp. 300 305. Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 370 377. Original Russian Text Copyright © 2005 by Tsybina, Dunaevsky, Belozersky, Zhuzhikov, Oppert, Elpidina. Originally published in Biochemistry (Moscow) On Line Papers in Press, as Manuscript BM04 310, February 20, 2005 ACCELERATED PUBLICATION 0006 2979/05/7003 0300 ©2005 Pleiades Publishing, Inc. Abbreviations: Bz) benzoyl; FPLC) fast performance liquid chromatography; For) formyl; PMSF) phenylmethylsulfonyl fluoride; pNA) p nitroanilide; TLCK) N α p tosyl L lysine chloromethyl ketone; TmT1) trypsin like proteinase from T. molitor larvae; E 64) L trans epoxysuccinyl L leucine amido(4 guanidine)butane; Z) N benzoyloxycarbonyl. * To whom correspondence should be addressed. Abstract-A new trypsin like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion exchange chromatography on DEAE Sephadex A 50 and gel filtration on Superdex 75. The isolated enzyme had molec ular mass of 25.5 kD and pI 7.4. The enzyme was also characterized by temperature optimum at 55°C, pH optimum at 8.5, and K m value of 0.04 mM (for hydrolysis of Bz Arg pNA). According to inhibitor analysis the enzyme is a trypsin like serine proteinase stable within the pH range of 5.0 9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N terminal amino acid sequence, IVGGSSI SISSVPXQIXLQY, shares 50 72% identity with other insect trypsin like proteinases, and 44 50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest. Digestive Proteinases of Yellow Mealworm (Tenebrio molitor