Purification and characterization of a 25 kDa cathepsin L-like protease from the hemocyte of coleopteran insect, Tenebrio molitor larvae

Kyung Suk Jang, Mi Young Cho, Hye Won Choi, Kang Moon Lee, Mi Hee Kim, Young Un Lee, Shoichiro Kurata, Shunji Natori, Bok Luel Lee

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Insect plasma protein is abundant in the hemolymph of holometabolous insect larvae and is used as a source of amino acids and energy for construction of adult structures during metamorphosis. In order to understand the mechanism of decomposition of larval plasma proteins by hemocyte protease, we tried to purify a cysteine protease from the hemocyte lysate by using Carbobenzoxy-L-Phenylalanyl-L-Arginine-4-Methyl-Coumaryl-7-Amide (Z-Phe-Arg-MCA) as substrate and to identify plasma proteins that are selectively susceptible to the purified protease. Here, we describe the purification and characterization of a cysteine protease that specifically hydrolyzes the plasma protein of the coleopteran insect, Tenebrio molitor, larvae. The molecular mass of this enzyme was 25 kDa, as determined by SDS-PAGE under reducing conditions. The amino acids sequence of its NH2-terminus was determined to be Leu-Pro-Gly-Gln-Ile-Asp-Trp-Arg-Asp-Lys-Gly. This sequence contained Pro, Asp, and Arg residues, conserved in many papain superfamily enzymes. The specific cysteine protease inhibitors, such as E-64 and leupetin, inhibited its hydrolytic activity. One plasma protein with a molecular mass of 48 kDa was selectively hydrolyzed within 3 h when the purified enzyme and plasma proteins were incubated in vitro. However, the 48 kDa protein was not hydrolyzed by the purified 25 kDa protease in the presence of E-64. Western blotting analysis at various developmental stages showed that the purified enzyme was detected at larvae, pupae, and adult stages, but not the embryo stage.

Original languageEnglish
Pages (from-to)364-369
Number of pages6
JournalJournal of Biochemistry and Molecular Biology
Volume31
Issue number4
Publication statusPublished - 1998 Jul 31
Externally publishedYes

Keywords

  • Cysteine protease
  • Hemocyte
  • Insect
  • Metamorphosis
  • Plasma proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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