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Assay Method Information |
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| | 5α-Reductase Activity Assay |
| Description: | The reaction mixture contained a final volume of 1 mL: 1 mM dithiothreitol, sodium phosphate buffer 40 mM, at pH 6.5 for human prostate, 2 mM NADPH, 2 nM [1,2,6,7-3H]T [Cabeza et al., Steroids 60:630-5]. The reaction in duplicate was started when it was added to the enzymatic fraction (500 μg protein in a volume of 80 μL) incubated at 37°C for 60 min [Hirosumi et al., J. Steroid Biochem. Mol. Biol., 52:357-63] and stopped by mixing with 1 mL of dichloromethane; this was considered as the end point. Incubation without tissue was used as a control. The mixture (incubation medium/dichloromethane) was agitated on a vortex for 1 min and the dichloromethane phase was separated and placed in another tube. This procedure was repeated four more times. The dichloromethane extract was evaporated to dryness under a nitrogen stream and suspended in 50 mL of methanol that was spotted on high-performance thin layer chromatography (HPTLC) Keiselgel 60 F254 plates. T and DHT were used as carriers, and were applied in different lanes on both lateral sides of the plates (T, T + DHT, and DHT). The plates were developed in chloroform-acetone, 9:1, and were air-dried; the chromatography was repeated twice more. The steroid carriers were detected using phosphomolybdic acid reagent (DHT) and with an ultraviolet (UV) lamp (254 nm) (T). After the plates were segmented into pieces of 1 cm each, these were cut off and the strips soaked in 5 mL of Ultima Gold (Packard). The radioactivity was determined in a scintillation counter (Packard Tri-Carb 2100 TR). The radioactivity content in the segment corresponding to T and DHT carriers was identified. Radioactivity with identical chromatographic behavior to the DHT standard was considered as the DHT transformation. Control incubations, chromatography separations, and identifications were carried out in the same manner as described above, except that the tubes did not contain tissue. |
| Affinity data for this assay | |
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