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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

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Abstract

The green rice leafhopper, Nephotettix cincticeps (Uhler), is an insect pest of rice and discharges β-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, β-glucosidases were eluted in three peaks (isozymes). These β-glucosidases were monomeric proteins of 58 kDa as estimated by SDS-PAGE and 62 kDa based on gel filtration. All of the purified β-glucosidase isozymes exhibited maximum activity for p-nitrophenyl β-glucoside (NPGlc) and p-nitrophenyl β-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 μM for NPGlc and 0.9 μM for NPGal. Among the oligosaccharide substrates examined, laminaribiose (Glc β1-3 Glc) was the most extensively hydrolyzed, sophorose (Glc β1-2 Glc) and cellobiose (Glc β1-4 Glc) were comparatively well hydrolyzed, and gentiobiose (Glc β1-6 Glc), lactose (Gal β1-4 Glc), laminaritriose, cellotriose and cellotetraose were poorly hydrolyzed. Among the glycoside substrates examined, salicin was considerably well hydrolyzed. β-Glucosidase was detected in the salivary sheaths by activity staining with a fluorescent substrate. The salivary β-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.

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Acknowledgments

We thank M. Watanabe for technical assistance, and K. Hashino and H. Niki for assistance with rearing and dissecting insects. We are grateful to Y. Tamura and T. Hasegawa for their valuable comments on the study. This work was partly supported by JSPS KAKENHI grant no. 22580063 and 25450075 to M.H.

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  1. Masatoshi Nakamura

    Present address: Genetic Resources Center, Kobuchizawa, Hokuto, Yamanashi, 408-0044, Japan

Authors and Affiliations

  1. Division of Insect Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8634, Japan

    Masatoshi Nakamura & Makoto Hattori

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  1. Masatoshi Nakamura
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  2. Makoto Hattori
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Correspondence to Makoto Hattori.

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Nakamura, M., Hattori, M. 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. Appl Entomol Zool 48, 489–497 (2013). https://doi.org/10.1007/s13355-013-0210-6

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  • DOI: https://doi.org/10.1007/s13355-013-0210-6

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