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- Title
Characterization of Cytochrome b5 in the Ascidian Polyandrocarpa misakiensis and Budding-Specific Expression1.
- Authors
Yubisu, Toshitsugu; Takahashi, Fumi; Takabayashi, Takashige; Fujiwara, Shigeki; Kawarnura, Kazuo
- Abstract
A cDNA for cytochrome b5 was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b5. The nucleotide sequence of the cDNA for the ascidian cytochrome b5 (Pmb5) consisted of about 1,800 base pairs including 5′- and 3′-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b5. A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding his-tidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escheriehia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b5 with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b5 reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome 65. 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 b5 in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.
- Subjects
PROTEIN analysis; CYTOCHROME b; DIGOXIGENIN; AMINO acid sequence; IMMUNOGLOBULINS
- Publication
Journal of Biochemistry, 2001, Vol 129, Issue 5, p709
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/oxfordjournals.jbchem.a002910