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Complex of digestive proteinases of Galleria mellonella caterpillars. Composition, properties, and limited proteolysis of Bacillus thuringiensis endotoxins

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Abstract

The complex of digestive proteinases in caterpillars of the greater wax moth Galleria mellonella was studied. Using chromogenic substrates and inhibitor analysis, it was found that serine proteinases play a key role in this complex. Three anionic and two cationic forms of trypsin and one anionic and one cationic form of chymotrypsin were identified by zymography in the midgut extract of G. mellonella. The most active trypsin was purified to electrophoretic homogeneity, and its N-terminal amino acid sequence was shown to be identical to that of mature trypsin from Plodia interpunctella. Midgut extract from G. mellonella was capable of processing Cry-proteins from Bacillus thuringiensis ssp. galleriae. Enzymes with tryptic and chymotryptic activities participate in this process, and activation of protoxin Cry9A is not the rate-limiting stage in the toxic action of this protein on the greater wax moth.

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Abbreviations

AM:

anterior midgut

Bz:

benzoyl

DFP:

diisopropylfluorophosphate

DNP:

dinitrophenyl

DTT:

dithiothreitol

E-64:

trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane

For:

formyl

Glp:

L-pyroglutamyl

PM:

posterior midgut

pNA:

p-nitroanilide

STI:

Kunitz soya trypsin inhibitor

Suc:

succinyl

TLCK:

tosyllysine chloromethyl ketone

TPCK:

tosylphenylalanine chloromethyl ketone

Z:

N-benzyloxycarbonyl

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Authors and Affiliations

  1. Scientific Research Institute for Genetics and Selection of Industrial Microorganisms, 1-yi Dorozhnyi Proezd 1, 117545, Moscow, Russia

    N. V. Bulushova, N. E. Kirillova, I. A. Zalunin & G. G. Chestukhina

  2. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991, Moscow, Russia

    E. N. Elpidina

  3. Biological Faculty, Lomonosov Moscow State University, 119991, Moscow, Russia

    D. P. Zhuzhikov & L. I. Lyutikova

  4. Centro de Investigaciones Biologicas, Ramiro de Maeztu 9, 28040, Madrid, Spain

    F. Ortego

Authors
  1. N. V. Bulushova
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  2. E. N. Elpidina
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  3. D. P. Zhuzhikov
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  4. L. I. Lyutikova
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  6. N. E. Kirillova
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  7. I. A. Zalunin
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  8. G. G. Chestukhina
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Corresponding author

Correspondence to E. N. Elpidina.

Additional information

Original Russian Text © N. V. Bulushova, E. N. Elpidina, D. P. Zhuzhikov, L. I. Lyutikova, F. Ortego, N. E. Kirillova, I. A. Zalunin, G. G. Chestukhina, 2011, published in Biokhimiya, 2011, Vol. 76, No. 5, pp. 713–723.

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Bulushova, N.V., Elpidina, E.N., Zhuzhikov, D.P. et al. Complex of digestive proteinases of Galleria mellonella caterpillars. Composition, properties, and limited proteolysis of Bacillus thuringiensis endotoxins. Biochemistry Moscow 76, 581–589 (2011). https://doi.org/10.1134/S0006297911050087

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  • DOI: https://doi.org/10.1134/S0006297911050087

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