5alpha-reductase in human embryonic kidney cell line HEK293: evidence for type II enzyme expression and activity.
Human 5alpha-reductase catalyses the last step in androgen biosynthesis, namely the reduction of testosterone (T) to the more potent androgen dihydrotestosterone (DHT). The enzyme is therefore considered to be an important drug target for androgen related diseases such as benign prostatic hyperplasia and prostate cancer. The present study displays evidence that the human embryonic kidney cell line HEK293 which is frequently used in recombinant target protein expression contains an endogenous 5alpha-reductase type II activity. After an incubation of 24 h 1 x 10(6) HEK293 cells converted 23% of the substrate 4-androstene-3,17-dione (7.5 nM) to the product 5alpha-androstane-3,17-dione. Reverse transcription polymerase chain reaction was carried out to identify the mRNA of the isoform responsible for the 5alpha-reductase activity. Only with type II specific primers a fragment with the predicted size was amplified, while with type I specific primers no band could be observed. An antiserum against human 5alpha-reductase type II was raised by immunizing a rabbit with a hemocyanin-conjugated peptide corresponding to amino acid 29 to 44 of the type II enzyme. Western blot analysis of different fractions of a HEK293 homogenate performed with this antiserum detected a band at 45 kDa in the nuclear and microsomal fraction corresponding to 5alpha-reductase type II protein.
Panter
Baerbel U
BU
Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbruecken, Germany.
Jose
Joachim
J
Hartmann
Rolf W
RW
D000735:Androstenedione / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D042944:Cholestenone 5 alpha-Reductase / Q000096:biosynthesis*; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000737:chemistry; D005043:Etiocholanolone / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D008861:Microsomes / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000735:Androstenedione; C006576:androstane-3,17-dione; D005043:Etiocholanolone; D010088:Oxidoreductases; D042944:Cholestenone 5 alpha-Reductase