Identification and partial characterization of midgut proteases in the lesser mulberry pyralid, Glyphodes pyloalis.
Proteolytic activities in digestive system extracts from the larval midgut of the lesser mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae), were analyzed using different specific peptide substrates and proteinase inhibitors. High proteolytic activities were found at pH 10.0 and a temperature of 50° C using azocasein as substrate. The trypsin was active in the pH range of 9.5- 12.0, with its maximum activity at pH 11.5. Ethylene diamine tetraacetic acid had the most inhibitory effect, and 44% inhibition was detected in the presence of this inhibitor. Phenyl methane sulfonyl floride and N-tosyl-L-phe chloromethyl ketone also showed considerable inhibition of larval azocaseinolytic activity, with 40.2 and 35.1% inhibition respectively. These data suggest that the midgut of larvae contains mainly metalloproteases and serine proteases, mainly chymotrypsin. The effect of several metal ions on the activity of proteases showed that NaCl, CaCl2, CoCl2 (5 and 10 mM), and MnCl2 (5mM) reduced the protease activity. The kinetic parameters of trypsin-like proteases using N-benzoyl-L-arg-p-nitroanilide as substrate indicated that the Km and Vmax values of trypsin in the alimentary canal were 50.5 ± 2.0 µM and 116.06 ± 1.96 nmol min(-1) mg(-1) protein, respectively. Inhibition assays showed only small amounts of cysteine proteases were present in the G. pyloalis digestive system. The midgut digestive protease system of G. pyloalis is as diverse as that of any of the other polyphagous lepidopteran insect species, and the midgut of larvae contains mainly metalloproteases. Moreover, serine proteases and chymotrypsin also play main roles in protein digestion. Characterization of the proteolytic properties of the digestive enzymes of G. pyloalis offers an opportunity for developing appropriate and effective pest management strategies via metalloproteases and chymotrypsin inhibitors.
1 Department of Biology, Faculty of sciences, University of Guilan, Rasht, Iran.
Atiyeh
A
Mahdavi
Mohammad
M
Ghadamyari
Reza H
RH
Sajedi
Mahbobeh
M
Sharifi
Behrooz
B
Kouchaki
D000818:Animals; D041981:Gastrointestinal Tract / Q000201:enzymology*; D007814:Larva / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D009036:Moths / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011480:Protease Inhibitors; D010447:Peptide Hydrolases; D014357:Trypsin