Biosynthesis of the antifreeze protein in the winter flounder, Pseudopleuronectus americanus: in vitro studies

Abstract

The serum of the winter flounder Pseudopleuronectus americanus contains one principal antifreeze protein of 10,000 daltons in the winter. In vitro incorporation with radioactive alanine in the flounder's liver showed the occurrence of one major radioactive component of 16,000 daltons on Sephadex G-75 columns in 0.05 M NH₄HCO₃ buffer, pH. 8.0, as well as in 8 M urea, 0.1 M tris-glycine buffer, pH 8.6, containing 50 mM β-mercaptoethanol. One major radioactive component was observed on SDS and urea tris-glycine diso gel electrophoresis. Upon mild tryptic digestion, this component yielded fragments having the same elution position on the gel filtration column as compared to authentic antifreeze protein. The synthesis of this 16,000 component in the fall (October-November) correspond closely with the serum antifreeze level and was absent from fish caught in the summer. However, the radioactivity in this in vitro biosynthetic product could not be converted to antifreeze protein using pulse chase experiments with puromycin, nor with cold alanine. -- An attempt was made to purify this antifreeze-like polypeptide from bulk materials. Amino acid analysis and enzymic peptide finger prints showed close structural similarity between this material and authentic antifreeze protein. -- It is concluded that this liver 16,000 dalton component represents the precursor protein for the antifreeze protein.