Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

• Published in: Journal of insect physiology Vol. 57; no. 4; pp. 462 - 468
• Main Authors: Soares-Costa, Andrea, Dias, Alcides B, Dellamano, Márcia, Paula, Fernando Fonseca Pereira de, Carmona, Adriana K, Terra, Walter R, Henrique-Silva, Flávio
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.04.2011
• Subjects: Animals; Cathepsin L; Cathepsin L - chemistry; Cathepsin L - metabolism; Cathepsin L-like proteinase; cathepsins; chemistry; chymotrypsin; Cysteine proteinase; cysteine proteinases; digestion; Digestive System; Digestive System - chemistry; Digestive System - enzymology; Digestive System Physiological Phenomena; enzymology; Insect Proteins; Insect Proteins - chemistry; Insect Proteins - metabolism; ion exchange chromatography; Kinetics; metabolism; midgut; parasitology; physiology; Plant resistance; Saccharum; Saccharum - parasitology; Sphenophorus; Sphenophorus levis; starch; Sugarcane; trypsin; Weevils; Weevils - chemistry; Weevils - enzymology; Weevils - physiology; SBTI; Cathepsin L-like proteinase; DTT; Z-FR48 MCA; E-64; Plant resistance; Cysteine proteinase; Z-RR-MCA; Sphenophorus levis; Z-LR-MCA; Sugarcane
• ISSN: 0022-1910; 1879-1611; 1879-1611
• DOI: 10.1016/j.jinsphys.2011.01.006

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with kcat/Km values of 20.0 +/- 1.1 micromolar-1 s-1 and 30.0 +/- 0.5 micromolar-1 s-1, respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane.