nitrophenols and adenosine-3--phosphate-5--phosphate

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.

Topics: Adenosine Diphosphate; Animals; Chromatography, DEAE-Cellulose; Diptera; Ecdysterone; Enzyme Inhibitors; Fat Body; Hemolymph; Larva; Naphthols; Nitrophenols; Phosphoadenosine Phosphosulfate; Photoaffinity Labels; Pupa; Sulfates; Sulfotransferases; Sulfur Radioisotopes

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cattle; Cornea; Electrophoresis; Epithelium; Metabolism; NAD; NADP; Nitrophenols; Nucleotides; Pyridines; Research; Sulfur Isotopes; Thioctic Acid; Tissue Extracts