In mammals, prodynorphin codes for three C-terminally extended forms of leu-enkephalin. This is not the case for the anuran amphibian, Bufo marinus. A combination of 3′RACE, RT-PCR and 5′RACE protocols was used to clone and characterize a prodynorphin cDNA from the brain of this amphibian that contained two met-enkephalin sequences. One met-enkephalin sequence was located at the N-terminal of Met5-dynorphin A(1–17), and the other met-enkephalin sequence was located in the N-terminal region of B. marinus prodynorphin in a position that aligned with a pentapeptide met-enkephalin site in mammalian proenkephalin. The latter B. marinus met-enkephalin sequence is flanked by sets of paired basic proteolytic cleavage sites. In addition to the extra met-enkephalin sequence and the Met5-dynorphin A(1–17) sequence, the B. marinus prodynorphin contained two C-terminally extended forms of leu-enkephalin [α-neo-endorphin and dynorphin B(1–13)]. In the toad precursor the α-neo-endorphin sequence is identical to human α-neo-endorphin. The B. marinus dynorphin B(1–13) sequence differs from human dynorphin B(1–13) by one amino acid (Thr12 vs. Val12). Steady-state analysis suggests that dynorphin B(1–13) and possibly α-neo-endorphin may be cleaved to yield leu-enkephalin as an end-product in the amphibian brain. Finally, the alignment of the extra met-enkephalin sequence in the N-terminal of B. marinus prodynorphin with the corresponding met-enkephalin site in mammalian proenkephalin adds support to the hypothesis that the prodynorphin gene arose as a duplication of the proenkephalin gene.
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