Figure 1

Identification of 7α-OH PREG and salmon P450_7α in the brain of chum salmon Oncorhynchus keta.

(a) HPLC profile of an unknown metabolite of PREG by using a reversed-phase column. The brain homogenates were incubated with [³H]PREG and the extracts were subjected to HPLC. The ordinate indicates the radioactivity measured in each HPLC fraction and the arrowheads indicate elution positions of standard steroids, PREG, 7α-OH PREG, 7β-OH PREG, stereoisomer of 7α-OH PREG, PROG and T. (b) GC-selected ion monitoring (SIM) mass trace of m/z 386 in the extract from brain homogenates. The arrowheads indicate the retention times of 7α-OH PREG and 7β-OH PREG. The m/z 386 ion is a diagnostic ion of 7α- and 7β-OH PREG^{17,18,19,20,21,22}. (c) 7α-OH PREG synthesis in COS-7 cells expressing the putative salmon Cyp7b. HPLC profile of [³H]7α-OH PREG extracted from COS-7 cells that were transfected with the putative salmon Cyp7b cDNA by pcDNA3.1/V5-His-TOPO and incubated with [³H]PREG. The ordinate indicates the radioactivity measured in each HPLC fraction and the arrowhead indicates the elution position of standard 7α-OH PREG. (d) HPLC profile of an extract from COS-7 cells that were transfected with the putative salmon Cyp7b cDNA and incubated with [³H]PREG in the presence of 10⁻⁴ M ketoconazole, an inhibitor of cytochrome P450s. (e) HPLC profile of an extract from COS-7 cells that were transfected with pcDNA3.1/V5-His-TOPO expression construct alone and incubated with [³H]PREG. (f) GC-MS analysis of 7α-OH PREG. GC-MS SIM trace (m/z 386) of the extract from COS-7 cells that were transfected with the putative salmon Cyp7b cDNA and incubated with PREG. The arrowhead shows the peak corresponding to 7α-OH PREG. The m/z 386 ion is a diagnostic ion of 7α-OH PREG^{17,18,19,20,21,22}. (g) GC-MS SIM trace (m/z 386) of the extract from nontransfected COS-7 cells. (h) The salmon brain expresses not only P450scc²⁹ but also P450₇α (present study) and produces 7α-OH PREG from cholesterol via PREG (present study).