Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression

A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the as cidian, Polyandrocarpa misakiensis, by a hybridization method involving a d igoxigenin-labeled cDNA probe of human soluble cytochrome b(5). The nucleot ide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a cod ing sequence of 405 base pairs. The amino acid sequence of 135 residues ded uced from the coding nucleotide sequence exhibited 54% identity and 76% sim ilarity to chicken cytochrome b(5). A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two hem e-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column ch romatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetri c peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH -cytochrome b(5) reductase, The low temperature spectrum of the dithionite- reduced form of the protein contained the split peaks at 551 and 555 nm, th is spectrum being very similar to that of mammalian liver cytochrome b(5). Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) i n the parents and juvenile adults were very low. This is the first report o n the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.