Purification and characterization of a 3,5,3'-L-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin.
We analyzed the nature of the thyroid hormone-binding proteins in bullfrog plasma using N-bromoacetyl-[125I]T3 as an affinity labeling probe. Polyacrylamide gel electrophoresis under nondenaturing conditions of the bullfrog N-bromoacetyl-[125I]T3-labeled plasma proteins revealed two proteins with specific binding to T3. A labeled protein that migrated faster than albumin (T-T3BP) was detected only in plasma obtained from tadpoles at stages earlier than, but not at the end of, metamorphic climax. Another protein that migrated more slowly than albumin appeared in the plasma at the late climax stage and was present in the adult stage. To study the function of T-T3BP during metamorphosis, it was purified from tadpole plasma to the single protein. The molecular mass of this protein was estimated to be 56 kilodaltons by gel filtration, but only 16 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the molecule comprised four identical subunits. The amino acid composition of T-T3BP and the amino acid sequence of its N-terminal portion were highly homologous with those of mammalian transthyretins. These molecular features indicate that T-T3BP is a homolog of mammalian transthyretins. However, in contrast to mammalian transthyretins, the affinity of bullfrog transthyretin for T3 was 360 times higher than that for T4. Scatchard analysis revealed that T-T3BP possessed a single class of T3-binding site, with a Kd of 0.67 nM at 0 C. These results suggest that bullfrog transthyretin may play an important role in transporting T3 in the blood during metamorphosis.
Yamauchi
K
K
Department of Biology, Faculty of Science, Shizuoka University, Japan.
Kasahara
T
T
Hayashi
H
H
Horiuchi
R
R
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000097:blood* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D007814:Larva / Q000378:metabolism*; D008565:Membrane Proteins / Q000097:blood* / Q000737:chemistry; D008969:Molecular Sequence Data; D011228:Prealbumin / Q000737:chemistry / Q000378:metabolism; D011892:Rana catesbeiana / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D013963:Thyroid Hormones; D014284:Triiodothyronine / Q000378:metabolism; D000097810:Thyroid Hormone-Binding Proteins
D000345:Affinity Labels; D000596:Amino Acids; D002352:Carrier Proteins; D008565:Membrane Proteins; D011228:Prealbumin; D013963:Thyroid Hormones; D014284:Triiodothyronine; D000097810:Thyroid Hormone-Binding Proteins