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A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae.
Biochimie. 2005 Aug; 87(8):771-9.B

Abstract

A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.

Authors+Show Affiliations

A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory, Moscow 119992, Russia. elp@belozersky.msu.ruNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

15885871

Citation

Elpidina, E N., et al. "A Chymotrypsin-like Proteinase From the Midgut of Tenebrio Molitor Larvae." Biochimie, vol. 87, no. 8, 2005, pp. 771-9.
Elpidina EN, Tsybina TA, Dunaevsky YE, et al. A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. Biochimie. 2005;87(8):771-9.
Elpidina, E. N., Tsybina, T. A., Dunaevsky, Y. E., Belozersky, M. A., Zhuzhikov, D. P., & Oppert, B. (2005). A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. Biochimie, 87(8), 771-9.
Elpidina EN, et al. A Chymotrypsin-like Proteinase From the Midgut of Tenebrio Molitor Larvae. Biochimie. 2005;87(8):771-9. PubMed PMID: 15885871.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. AU - Elpidina,E N, AU - Tsybina,T A, AU - Dunaevsky,Y E, AU - Belozersky,M A, AU - Zhuzhikov,D P, AU - Oppert,B, Y1 - 2005/04/19/ PY - 2004/12/20/received PY - 2005/02/08/accepted PY - 2005/5/12/pubmed PY - 2005/11/15/medline PY - 2005/5/12/entrez SP - 771 EP - 9 JF - Biochimie JO - Biochimie VL - 87 IS - 8 N2 - A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes. SN - 0300-9084 UR - https://www.unboundmedicine.com/medline/citation/15885871/A_chymotrypsin_like_proteinase_from_the_midgut_of_Tenebrio_molitor_larvae_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0300-9084(05)00093-3 DB - PRIME DP - Unbound Medicine ER -