Purification and Characterization of Prophenoloxidase from the Hemolymph of Coleopteran Insect, Holotrichia diomphalia Larvae

Tae Hyuk Kwon, So Young Lee, Ji Hye Lee, Jae Sue Choi, Shun Ichiro Kawabata, Sadaaki Iwanaga, Bok Luel Lee

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Abstract

Prophenoloxidase (pro-PO), a precursor of phenol oxidase (PO), was purified from the hemolymph of coleopteran Holotrichia diomphalia larvae. The enzyme was purified to apparent homogeneity, in six steps of chromatography, using Sephadex G-100, CM-52, Dextran-sulfate Sepharose CL-6B, Phenyl Sepharose CL-4B, Sephacryl S-200, and a Mono-Q column. The preparation exhibited a single band on SDS-PAGE. The proenzyme had a molecular weight of 158 kDa, as estimated by gel filtration. On SDS-PAGE under reducing conditions, it gave a 79 kDa band, indicating that it forms a dimer of the 79 kDa protein. On the other hand, the purified pro-PO gave two well-separated peaks, named proPO-1 and pro-PO-2, on reverse-phase HPLC. Amino acid compositions of both proteins were indistinguishable, thereby suggesting the presence of an allelic variant or an isoprotein. On dextran-sulfate Sepharose CL-6B chromatography, a fraction containing prophenoloxidase activating enzyme(s) (PPAE fraction), free from pro-PO, was also separated. In reconstitution experiments, the activation of purified pro-PO by PPAE fraction was observed in the presence of 5 mM Ca2+, with a specific limited proteolysis. The NH2-terminal sequence of generated PO was determined to be NH2-Phe-Gly-Glu-Asp-Asp-. The activated PO oxidized o-diphenols but did not oxidize mono-phenol and p-diphenol substrates. The purified pro-PO was not activated by trypsin, α-chymotrypsin, and SDS.

Original languageEnglish
Pages (from-to)90-97
Number of pages8
JournalMolecules and cells
Volume7
Issue number1
Publication statusPublished - Feb 28 1997

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All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

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