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Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera).
Arch Insect Biochem Physiol. 2015 Mar; 88(3):192-202.AI

Abstract

We determined some biochemical properties of Oulema melanopus larval gut proteases. We found adult midgut enzyme preparations yielded results similar to whole-larval preparations, permitting studies of the very small whole-larval preparations. Protein preparations were analyzed using FITC-casein as a substrate. Acidic pH is optimal for proteolytic activity (range 3.0-4.0). Cysteine protease activity increased at acidic pH and in the presence of β-mercaptoethanol. Protease activities at all pH values were maximal at 45°C. Enzyme activity in larval preparations was inhibited by addition of Fe(2+) , Ca(2+) , Mg(2+) , Zn(2+) , and K(+) (10 mM). Fe(2+) and Zn(2+) significantly decreased enzyme activity at all pH values, Ca(2+) and Mg(2+) at pH 6.2 and Mg(2+) at pH 4.0. Inhibitors, including pepstatin A, showed the greatest inhibition at pH 4.0; phenylmethylsulfonyl fluoride, N-p-tosyl-l-phenylalanine chloromethyl ketone at pH 6.2; and phenylmethylsulfonyl fluoride, Nα -tosyl-l-lysine chloromethyl ketone hydrochloride, N-p-tosyl-l-phenylalanine chloromethyl ketone, trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane at pH of 7.6. Inhibition assays indicated that cysteine, aspartyl (cathepsin D), serine (trypsin, chymotrypsin-like) proteases and metalloproteases act in cereal leaf beetle digestion.

Authors+Show Affiliations

Department of Agrophages´ Forecasting Methods and Agricultural Economics, Institute of Plant Protection - National Research Institute, Poznań, Poland.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25580929

Citation

Wielkopolan, Beata, et al. "Identification and Partial Characterization of Proteases in Larval Preparations of the Cereal Leaf Beetle (Oulema Melanopus, Chrysomelidae, Coleoptera)." Archives of Insect Biochemistry and Physiology, vol. 88, no. 3, 2015, pp. 192-202.
Wielkopolan B, Walczak F, Podleśny A, et al. Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera). Arch Insect Biochem Physiol. 2015;88(3):192-202.
Wielkopolan, B., Walczak, F., Podleśny, A., Nawrot, R., & Obrępalska-Stęplowska, A. (2015). Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera). Archives of Insect Biochemistry and Physiology, 88(3), 192-202. https://doi.org/10.1002/arch.21223
Wielkopolan B, et al. Identification and Partial Characterization of Proteases in Larval Preparations of the Cereal Leaf Beetle (Oulema Melanopus, Chrysomelidae, Coleoptera). Arch Insect Biochem Physiol. 2015;88(3):192-202. PubMed PMID: 25580929.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera). AU - Wielkopolan,Beata, AU - Walczak,Felicyta, AU - Podleśny,Andrzej, AU - Nawrot,Robert, AU - Obrępalska-Stęplowska,Aleksandra, Y1 - 2015/01/12/ PY - 2015/1/13/entrez PY - 2015/1/13/pubmed PY - 2015/9/29/medline KW - aspartyl protease KW - cereal leaf beetle (Oulema melanopus) KW - cysteine protease KW - metalloprotease KW - protease inhibitors KW - proteases KW - serine protease SP - 192 EP - 202 JF - Archives of insect biochemistry and physiology JO - Arch Insect Biochem Physiol VL - 88 IS - 3 N2 - We determined some biochemical properties of Oulema melanopus larval gut proteases. We found adult midgut enzyme preparations yielded results similar to whole-larval preparations, permitting studies of the very small whole-larval preparations. Protein preparations were analyzed using FITC-casein as a substrate. Acidic pH is optimal for proteolytic activity (range 3.0-4.0). Cysteine protease activity increased at acidic pH and in the presence of β-mercaptoethanol. Protease activities at all pH values were maximal at 45°C. Enzyme activity in larval preparations was inhibited by addition of Fe(2+) , Ca(2+) , Mg(2+) , Zn(2+) , and K(+) (10 mM). Fe(2+) and Zn(2+) significantly decreased enzyme activity at all pH values, Ca(2+) and Mg(2+) at pH 6.2 and Mg(2+) at pH 4.0. Inhibitors, including pepstatin A, showed the greatest inhibition at pH 4.0; phenylmethylsulfonyl fluoride, N-p-tosyl-l-phenylalanine chloromethyl ketone at pH 6.2; and phenylmethylsulfonyl fluoride, Nα -tosyl-l-lysine chloromethyl ketone hydrochloride, N-p-tosyl-l-phenylalanine chloromethyl ketone, trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane at pH of 7.6. Inhibition assays indicated that cysteine, aspartyl (cathepsin D), serine (trypsin, chymotrypsin-like) proteases and metalloproteases act in cereal leaf beetle digestion. SN - 1520-6327 UR - https://www.unboundmedicine.com/medline/citation/25580929/Identification_and_partial_characterization_of_proteases_in_larval_preparations_of_the_cereal_leaf_beetle__Oulema_melanopus_Chrysomelidae_Coleoptera__ DB - PRIME DP - Unbound Medicine ER -