Identification and purification of sulfotransferases for 20-hydroxysteroid from the larval fat body of a fleshfly, Sarcophaga peregrina.
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 microM), 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.
Matsumoto
Eiko
E
Kyoritsu College of Pharmacy, Minato-ku, Tokyo 105-8512, Japan.
Matsui
Michio
M
Tamura
Hiro-omi
HO
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D004175:Diptera / Q000201:enzymology* / Q000254:growth & development; D004441:Ecdysterone / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005216:Fat Body / Q000737:chemistry / Q000201:enzymology*; D006458:Hemolymph / Q000378:metabolism; D007814:Larva / Q000737:chemistry / Q000201:enzymology; D009284:Naphthols / Q000302:isolation & purification / Q000378:metabolism; D009596:Nitrophenols / Q000494:pharmacology; D010724:Phosphoadenosine Phosphosulfate / Q000031:analogs & derivatives / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism; D011679:Pupa / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013462:Sulfur Radioisotopes
D004791:Enzyme Inhibitors; D009284:Naphthols; D009596:Nitrophenols; D019920:Photoaffinity Labels; D013431:Sulfates; D013462:Sulfur Radioisotopes; D010724:Phosphoadenosine Phosphosulfate; D004441:Ecdysterone; C015802:2,6-dichloro-4-nitrophenol; D000244:Adenosine Diphosphate; C010840:adenosine 3'-phosphate-5'-phosphate; D015238:Sulfotransferases; C028405:2-naphthol