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 molecular mass of 25.5 kD and pI7.4. 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, IVGGSSISISSVPXQIXLQY, 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.
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Abbreviations
- Bz:
-
benzoyl
- FPLC:
-
fast performance liquid chromatography
- For:
-
formyl
- PMSF:
-
phenylmethylsulfonyl fluoride
- pNA:
-
p-nitroanilide
- TLCK:
-
Nα-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
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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.
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Tsybina, T.A., Dunaevsky, Y.E., Belozersky, M.A. et al. Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: Purification and characterization of a trypsin-like proteinase. Biochemistry (Moscow) 70, 300–305 (2005). https://doi.org/10.1007/s10541-005-0115-2
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DOI: https://doi.org/10.1007/s10541-005-0115-2