Purification of β-glucosidase from the salivary glands of the green rice leafhopper, Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae), and its detection in the salivary sheath

The green rice leafhopper, Nephotettix cincticeps (Uhler), is an insect pest of rice and discharges beta-glucosidase (EC 3.2.1.21) from its salivary glands during feeding. To investigate the biological function of this enzyme, we purified it from the heads of 18,000 adult females by acetone precipitation and a series of chromatography steps: gel filtration, cation-exchange chromatography, metal-affinity chromatography and hydrophobic interaction chromatography. During cation-exchange chromatography, beta-glucosidases were eluted in three peaks (isozymes). These beta-glucosidases were monomeric proteins of 58 kDa as estimated by SDS-PAGE and 62 kDa based on gel filtration. All of the purified beta-glucosidase isozymes exhibited maximum activity for p-nitrophenyl beta-glucoside (NPGlc) and p-nitrophenyl beta-galactopyranoside (NPGal) at pH 5.5 and 5.0, respectively. There was no significant difference in substrate specificity among the three isozymes. The Km values were estimated to be 0.13 mu M for NPGlc and 0.9 mu M for NPGal. Among the oligosaccharide substrates examined, laminaribiose (Glc beta 1-3 Glc) was the most extensively hydrolyzed, sophorose (Glc beta 1-2 Glc) and cellobiose (Glc beta 1-4 Glc) were comparatively well hydrolyzed, and gentiobiose (Glc beta 1-6 Glc), lactose (Gal beta 1-4 Glc), laminaritriose, cellotriose and cellotetraose were poorly hydrolyzed. Among the glycoside substrates examined, salicin was considerably well hydrolyzed. beta-Glucosidase was detected in the salivary sheaths by activity staining with a fluorescent substrate. The salivary beta-glucosidase of N. cincticeps may be involved in the hydrolysis of a phenol glucoside present in the saliva, which is a step in the solidification of gelling saliva to form salivary sheaths.