TY - JOUR
AU - TAMURA, Hiro-omi
AB - Abstract Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC50=0.61 μM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3′-phosphoadenosine 5′-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [35S]-3′-phosphoadenosine 5′-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina. 20-hydroxyecdysone, sulfotransferase, fly, Sarcophaga peregrina References 1) Weinshilboum, R., and Otterness, D., Sulfotransferase enzymes. In “Drug Metabolism and Toxicity”, ed. Kauffman, F. C., Springer-Verlag, Berlin, pp. 45-78 (1994). 2) Falany, C. N., Enzymology of human cytosolic sulfotransferases. FASEB J., 11, 206-216 (1997). 3) Nagata, K., and Yamazoe, Y., Pharmacogenetics of sulfotransferase. Ann. Rev. Pharmacol. Toxicol., 40, 159-176 (2000). 4) Smith, J. N., Comparative detoxication.—4. Ethreal sulphate and glucoside conjugation in insects. Biochem. J., 92, 127-131 (1955). 5) Yang, R. S. H., and Wilkinson, C. F., Enzymatic suphation of p-nitrophenol and steroids by larval gut tissues of the southern armyworm (Prodenia eridania cramer). Biochem. J., 130, 487-493 (1972). 6) Slade, M., and Wilkinson, C. F., Degradation and conjugation of Cecropia juvenile hormones by the southern armyworm (Prodenia eridania). Comp. Biochem. Physiol., 49B, 99-103 (1974). 7) Koolman, J., Hoffmann, J. A., and Karlson, P., Sulphate esters as inactivation products of ecdysone in Locusta migratoria. Hoppe-Seyler's Z. Physiol. Chem., 354, 1043-1048 (1973). 8) Enderle, U., and Koolman, J., Ecdysteroid sulphotransferase: analysis of the enzyme in vitro. Biochem. Soc. Trans., 9, 156P (1981). 9) Shampengtong, L., and Wong, K. P., An in vitro assay of 20-hydroxyecdysone sulfotransferase in the mosquito, Aedes togoi. Insect Biochem., 19, 191-196 (1989). 10) Homma, H., Kamakura, M., Nakagome, I., and Matsui, M., Purification of a rat phenol sulphotransferase (P-STG) with the aid of guanidine hydrochloride treatment. Chem. Pharm. Bull., 39, 3307-3312 (1991). 11) Foldes, A., and Meek, J. L., Rat brain phenol-sulfotransferase; Partial purification and some properties. Biochim. Biophys. Acta, 327, 365-374 (1973). 12) Tamura, H., Miyawaki, A., Yoneshima, H., Mikoshiba, K., and Matsui, M., Molecular cloning, expression and characterization of a phenol sulfotransferase cDNA from mouse intestine. Biol. Pharm. Bull., 22, 234-239 (1999). 13) Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685 (1970). 14) Seah, V. M., and Wong, K. P., 2-6-Dichloro-4-nitropheenol (DCNP), an alternative inhibitor of phenolsulfotransferase. Biochem. Pharmacol., 47, 1743-1749 (1994). 15) Ogura, K., Sohtome, T., Sugiyama, A., Okuda, H., Hiratsuka, A., and Watabe, T., Rat liver cytosolic hydroxysteroid sulfotransferase (sulfotransferase a) catalyzing the formation of reactive sulfate esters from carcinogenic polycyclic hydroxymethylarenes. Mol. Pharmacol., 37, 848-854 (1990). 16) Tamura, H., Morioka, Y., Homma, H., and Matsui, M., Construction and expression of chimeric rat liver hydroxysteroid sulfotransferase isozymes. Arch. Biochem. Biophys., 341, 309-314 (1997). 17) Okuda, H., Nojima, H., Watanabe, N., and Watabe, T., Sulphotransferase-mediated activation of the carcinogen 5-hydroxymethylchrysene: species and sex differences in tissue distribution of the enzyme activity and a possible participation of hydroxysteroid sulphotransferses. Biochem. Pharmacol., 38, 3003-3009 (1989). PDF This content is only available as a PDF. © 2003 by Japan Society for Bioscience, Biotechnology, and Agrochemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) © 2003 by Japan Society for Bioscience, Biotechnology, and Agrochemistry
TI - Identification and Purification of Sulfotransferases for 20-Hydroxysteroid from the Larval Fat Body of a Fleshfly, Sarcophaga peregrina
JO - Bioscience Biotechnology and Biochemistry
DO - 10.1271/bbb.67.1780
DA - 2003-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/identification-and-purification-of-sulfotransferases-for-20-r3B2acegWt
SP - 1780
EP - 1785
VL - 67
IS - 8
DP - DeepDyve
ER -