Isolation, characterization and structural studies of the antifreeze polypeptides from ocean pout, Macrozoarces americanus

Li, Xiaomao (1985) Isolation, characterization and structural studies of the antifreeze polypeptides from ocean pout, Macrozoarces americanus. Masters thesis, Memorial University of Newfoundland.

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Abstract

Antifreeze polypeptides (AFP) were separated successfully from the serum of ocean pout Macroxoarces americanus using Sephadex G-75 gel-filtration. QAE-Sephadex and SP-Sephadex ion-exchange chromatography and reverse phase HPLC. -- It was found that the ocean pout AFP consists of a multiple family of at least twelve active components of nearly identical size (about 6,000 daltons), which can be classified into two separate groups, named QAE and SP on the basis of their amino acid compositions and their behaviour on ion-exchange chromatography. -- A partial amino acid sequence of SP-1-A, one of the major ocean pout AFP, was established up to 41 residues from its chymotryptic peptides using a protein sequenator. SP-1-A was compared with two other major components, namely SP-1-B and SP-1-C, by tryptic and chymotryptic peptide mapping and amino acid analyses. They showed overall structural similarities with minor differences. -- A cDNA coding for the biosynthetic precursor of one of the ocean pout AFP was isolated and purified from the plasmid pBR 322 in E. Coli HB101. It was then cloned into phage M13 mp8. The sequence of the cDNA was determined by the dideoxy chain termination method. It had untranslated regions at both 3' and 5', and a coding region for a signal peptide containing 22 amino acid residues and a mature polypeptide containing 65 residues. The sequence of the mature polypeptide matched the protein data from both SP-1-B and SP-1-C. The results from both protein chemistry and molecular biology showed that SP-1-A, SP-1-B and SP-1-C have very similar amino acid compositions and sequences with only minor differences. For example, SP-1-A contains Ile and Ala at positions 53 and 62, while both SP-1-B and SP-1-C contain respectively Leu and Val at the same positions. SP-1-C has an additional Gly at its C-terminal compared to both SP-1-A and SP-1-B. -- The amino acid analyses showed that SP-1-A, SP-1-B and SP-1-C contain fourteen types of amino acids with modest contents of Ala and no half-cystine. There is no obvious repeating structure in ocean pout AFP. These results confirm that ocean pout AFP represent a new type of fish antifreeze proteins. -- Ocean pout AFP from individual fish caught in both winter and summer from Newfoundland, and in winter months from New-Brunswick were analyzed on reverse phase HPLC. The results did not show any significant populational or seasonal polymorphisms.

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