Hostname: page-component-78c5997874-v9fdk Total loading time: 0 Render date: 2024-11-10T16:10:23.744Z Has data issue: false hasContentIssue false
  • English
  • Français

Detoxification of cyanide in insects. I. Purification and some properties of rhodanese from the gut of the variegated grasshopper Zonocerus variegatus (Orthoptera: Pyrgomorphidae)

Published online by Cambridge University Press:  01 September 2013

Igue Udoka Bessie  and
Femi Kayode Agboola
Igue Udoka Bessie
Affiliation:
Department of Biochemistry, Obafemi Awolowo University, Ile-Ife 220005, Nigeria
Femi Kayode Agboola*
Affiliation:
Department of Biochemistry, Obafemi Awolowo University, Ile-Ife 220005, Nigeria
*
*E-mail: fkagbo@oauife.edu.ng
Get access

Abstract

The purification and characterization of rhodanese, an enzyme that catalyses the detoxification of cyanide, from the gut of the variegated grasshopper (Zonocerus variegatus L.) were carried out to understand the biochemical basis of the survival of this grasshopper living on cyanogenic plants such as cassava. All experiments, including enzyme assay, were carried out at room temperature and all buffers contained 10 mm sodium thiosulphate to stabilize the enzyme. Grasshoppers were caught alive from a cassava farm within the locality and kept frozen until analysis. Each grasshopper was dissected and the gut was removed quickly. Approximately 102 g of the gut were homogenized in three volumes of 0.1 m acetate glycine buffer (pH 7.8) containing ɛ-amino-n-caproic acid. The supernatant was collected by centrifugation at 12,000 rpm, for 30 min at 40 °C. The enzyme was purified to homogeneity by a combination of procedures such as ammonium sulphate precipitation, ion-exchange chromatography (CM-Sephadex and DEAE-Sephadex), gel filtration (Sephadex G-75) and Agarose-Blue affinity chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and non-SDS–PAGE were used to ascertain the purity of the enzyme. The native and subunit molecular weights of the enzyme were determined by gel filtration on a Bio-Gel P-200 column and SDS–PAGE, respectively. Kinetic parameters were determined by using varying concentrations of one of the substrates at a fixed concentration of the other, and vice versa. Furthermore, the effects of temperature, pH and cations on the activity of the enzyme were investigated. The purified enzyme had a specific activity of 51.7 μmol thiocyanate formed/ml/min/mg protein (U/mg protein) with a yield of about 29%. The apparent molecular weight of the enzyme estimated by Sephadex G-75 gel filtration was 35,400 ± 482 Da and its subunit molecular weight determined by SDS–PAGE was 33,000 ± 212 Da. The Km values of KCN and Na2S2O3 were found to be 29.63 ± 02.87 and 26 ± 03.04 mm, respectively. An optimum pH and temperature of 7.0 and 35 °C, respectively, were obtained for the enzyme. The results of enzyme inhibition showed that the activity of the enzyme was not affected by NH4Cl, MgCl2, CoCl2, CaCl2, MnCl2, NiCl2 and SnCl2, but inhibited by ZnCl2 and BaCl2. In conclusion, these results suggest that the survival of Z. variegatus depends on the presence of the enzyme rhodanese, which shows high activity and has suitable kinetic properties in the gut of the grasshopper that feeds mainly on cassava leaves which are cyanogenic.

Keywords

rhodanesecyanidegrasshopperZonocerus variegatusgutinsect
Type
Research Papers
Information
International Journal of Tropical Insect Science , Volume 33 , Issue 3 , September 2013 , pp. 153 - 162
Copyright
Copyright © icipe 2013 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Agboola, F. K. and Okonji, R. E. (2004) Presence of rhodanese in the cytosolic fraction of the fruit bat (Eidolon helvum) liver. Journal of Biochemistry and Molecular Biology 37, 275–281.Google Scholar
Akinsiku, O. T., Agboola, F. K., Kuku, A. and Afolayan, A. (2010) Physicochemical and kinetic characteristics of rhodanese from the liver of African catfish Clarias gariepinus Burchell in Asejire lake. Fish Physiology and Biochemistry 36, 573–586.CrossRefGoogle ScholarPubMed
Al-Qarawi, A. A., Mousa, H. M. and Ali, B. H. (2001) Tissue and intracellular distribution of rhodanese and mercaptopyruvate sulphurtranferase in ruminants and birds. Veterinary Research 32, 63–70.Google Scholar
Andrews, P. (1964) Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochemical Journal 91, 222–233.CrossRefGoogle ScholarPubMed
Anosike, E. O. and Ugochukwu, E. N. (1981) Characterization of rhodanese from cassava leaves and tubers. Journal of Experimental Botany 32, 1021–1027.CrossRefGoogle Scholar
Ballantyne, B. (1987) Toxicology of cyanides, pp. 41–126. In Clinical and Experimental Toxicology of Cyanides (edited by Ballantyne, B. and Marrs, T. C.). John Wright Publishing, Bristol.Google Scholar
Blumenthal, K. M. and Heinrikson, R. L. (1971) Structural studies of bovine liver rhodanese: I. Isolation and characterization of two active forms of the enzyme. Journal of Biological Chemistry 246, 2430–2437.Google Scholar
Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.Google Scholar
Butler, G. W., Barley, R. W. and Kennedy, L. D. (1965) Studies on the glucoside ‘linamarase’. Phytochemistry 4, 369–381.Google Scholar
Chew, M. Y. and Boey, C. G. (1972) Rhodanese of tapioca leaf. Phytochemistry 11, 167–169.Google Scholar
Claus, E., Tyler, V. E. and Brady, L. R. (1970) Pharmacognosy, 6th edn.Lea & Febiger, Philadelphia. 518 pp.Google Scholar
Cooke, R. D. (1978) An enzymatic assay for the total cyanide content of cassava (Manihot esculenta Crantz). Journal of the Science of Food and Agriculture 29, 345–352.Google Scholar
Conn, E. E. (1979) Cyanide and cyanogenic glycosides, pp. 387–412. In Herbivores: Their Interaction with Secondary Plant Metabolites (edited by Rosenthal, G. A. and Janzen, D. H.). Academic Press, New York.Google Scholar
De Bruijn, G. H. (1973) The cyanogenic character of cassava (Manihot esculenta), pp. 43–48. In Chronic Cassava Toxicity. Proceedings of an Interdisciplinary Workshop. 29–30 January 1973, London (edited by Nestel, B. and MacIntyre, R.). International Development Research Centre, Ottawa.Google Scholar
Ezzi, M. I., Pascual, J. A., Gould, B. J. and Lynch, J. M. (2003) Characterisation of the rhodanese enzyme in Trichoderma spp. Enzyme and Microbial Technology 32, 629–634.Google Scholar
Himwich, W. A. and Saunders, J. B. (1948) Enzymatic conversion of cyanide to thiocyanate. American Journal of Physiology 153, 348–354.Google Scholar
Jarabak, R. and Westley, J. (1978) Steady-state kinetics of 3-mercaptopyruvate sulphurtransferase from bovine kidney. Archives of Biochemistry and Biophysics 185, 458–465.CrossRefGoogle Scholar
Jones, D. A. (1998) Why are so many food plants cyanogenic? Phytochemistry 47, 155–162.Google Scholar
Keilin, D. (1929) Cytochrome and respiratory enzymes. Proceedings of the Royal Society of London Series B 104, 206–252.Google Scholar
Lang, K. (1933) Die Rhodanidebildung im Tierkörper. Biochemische Zeitschrift 259, 243–256.Google Scholar
Lee, C. H., Hwang, J. H., Lee, Y. S. and Cho, K. S. (1995) Purification and characterization of mouse liver rhodanese. Journal of Biochemistry and Molecular Biology 28, 170–176.Google Scholar
Lieske, C. N., Clark, C. R., Zoeffel, L. D., von Tersch, R. L., Lowe, J. R., Smith, C. D., Broomfield, C. A., Baskin, S. I. and Maxwell, D. M. (1996) Temperature effects in cyanolysis using elemental sulfur. Journal of Applied Toxicology 16, 171–175.Google Scholar
Lindroth, R. L. (1991) Differential toxicity of plant allelochemicals to insects: roles of enzymatic detoxication systems, pp. 1–33. In Insect–Plant Interaction (edited by Bernays, E.). CRC Press, Boca Raton.Google Scholar
Meister, A. (1953) Cytosolic mercaptopyruvate sulphurtransferase is evolutionarily related to mitochondrial rhodanese. Federation Proceedings 12, 245.Google Scholar
Modder, W. W. D. (1994) Control of the variegated grasshopper Zonocerus variegatus (L.) on cassava. African Crop Science Journal 2, 391–406.Google Scholar
Nagahara, N., Ito, T. and Minami, M. (1999) Mercaptopyruvate sulphurtransferase as a defense against cyanide toxication: molecular properties and mode of detoxification. Histology and Histopathology 14, 1277–1286.Google ScholarPubMed
Nagahara, N., Okazaki, T. and Nishino, T. (1995) Cytosolic mercaptopyruvate sulphurtransferase is evolutionarily related to mitochondrial rhodanese. Striking similarity in active site amino acid sequence and the increase in the mercaptopyruvate sulfurtransferase activity of rhodanese by site-directed mutagenesis. Journal of Biological Chemistry 270, 16230–16235.Google Scholar
Nartey, F. (1968) Studies on cassava, Manihot utilissima Pohl. L. cyanogenesis: the biosynthesis of linamarin and lotaustralin in etiolated seedlings. Phytochemistry 7, 1307–1312.Google Scholar
Nicol, C. M. Y., Assadsolimani, D. C. and Langewald, J. (1995) Caelifera: short-horned grasshoppers and locusts, pp. 233–248. In Neem Tree Azadirachta indica A. Juss. and Other Meliaceous Plants (edited by Schmutterer, H.). VCH, Weinheim.Google Scholar
Ogunlabi, O. O. and Agboola, F. K. (2007) A soluble β-cyanoalanine synthase from the gut of the variegated grasshopper Zonocerus variegatus (L.). Insect Biochemistry and Molecular Biology 37, 72–79.Google Scholar
Oke, O. L. (1979) Some aspects of the role of cyanogenic glycosides in nutrition. World Review of Nutrition and Dietetics 33, 70–103.CrossRefGoogle ScholarPubMed
Osuntokun, B. O. (1981) Cassava diet, chronic cyanide intoxication and neuropathy in the Nigerian Africans. World Review of Nutrition and Dietetics 36, 141–173.Google Scholar
Ploegman J. H., Drent G., Kalk K. H. and Hoi W. G. (1978) Structure of bovine liver rhodanese. I. Structure determination at 2.5 Å resolution and a comparison of the conformation and sequence of its two domains. Journal of Molecular Biology 123, 557–594.Google Scholar
Roy, A. B. and Trudinger, P. A. (1970) The Biochemistry of Inorganic Compounds of Sulphur. Cambridge University Press, Cambridge. 416 pp.Google Scholar
Russell J., Weng L., Kein P. S. and Heinrikson R. L. (1978) The covalent structure of bovine liver rhodanese. Journal of Biological Chemistry 253, 8102–8108.Google Scholar
Sorbo, B. H. (1951) On the properties of rhodanese. Acta Chemica Scandinavica 5, 724–726.Google Scholar
Sorbo, B. H. (1953a) Crystalline rhodanese. Acta Chemica Scandinavica 7, 1129–1136.CrossRefGoogle Scholar
Sorbo B. H. (1953b) Crystalline rhodanese. Enzyme catalyzed reaction. Acta Chemical Scandinavia 7, 1137–1145.Google Scholar
Sorbo, B. H. (1955) Rhodanese. Methods in Enzymology 2, 334–337.Google Scholar
Stokinger H. E. (1981) Industrial Hygiene and Toxicology, pp. 1493–2060. In Hygiene and Toxicology, 3rd edn, vol. 2A (edited by C. D. Clayton and F. E. Clayton). John Wiley and Sons, New York.Google Scholar
Tanka, D. and Gatai, K. (1983) Histochemical detection of thiosulphate sulphurtransferase (rhodanese) activity. Histochemistry 77, 285–288.Google Scholar
Tomati, U., Giovanni, G., Anni, S., Dupres, S. and Cannella, C. (1976) NADH: nitrate reductase activity restoration by rhodanese. Phytochemistry 15, 597–598.Google Scholar
Volini, M., DeToma, F. and Westley, J. (1967) Dimeric structure and zinc content of bovine liver rhodanese. Journal of Biological Chemistry 242, 5220–5225.Google Scholar
Volini M. and Wang S. F. (1978) Conformational stabilization of enzymes in covalent catalysis. Archives of Biochemistry and Biophysics 187, 163–169.CrossRefGoogle Scholar
Wang, S.-F. and Volini, M. (1968) Studies on the active site of rhodanese. Journal of Biological Chemistry 243, 5465–5470.Google Scholar
Warburg, O. (1911) Inhibition of the action of prussic acid in living cells. Zeitschrift für Physiologische Chemie (Journal of Physiological Chemistry) 76, 331–346.Google Scholar
Weber, K. and Osborn, M. (1975) Protein and sodium dodecyl sulphate: molecular weight determination on polyacrylamide gel and related procedures, pp. 179–223. In The Proteins Vol. I (edited by Neurath, H. and Hill, R. L.). Academic Press, New York.Google Scholar
Westley, J. (1973) Rhodanese. Advances in Enzymology and Related Areas of Molecular Biology 39, 327–368.Google Scholar
Westley, J. (1980) Rhodanese and the sulphane pool, pp. 245–259. In Enzymatic Basis of Detoxification Vol. II (edited by Jacoby, W. B.). Academic Press, New York.Google Scholar
Youdeowei, A. (1974) The Dissection of the Variegated Grasshopper, Zonocerus variegatus (L.). Oxford University Press, Ibadan. 101 pp.Google Scholar