A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis.
A trypsin inhibitor from Ciona intestinalis, present throughout the animal, was purified by ion-exchange chromatography followed by four HPLC steps. By MS the molecular mass of the native form was determined to be 6675 Da. The N-terminal amino acid sequence was determined by protein sequencing, but appeared to be partial because the theoretical molecular mass of the protein was 1101 Da too low. Thermolysin treatment gave rise to several fragments each containing a single disulphide bridge. By sequence analysis and MS intramolecular disulphide bridges could unequivocally be assigned to connect the pairs Cys4-Cys37, Cys8-Cys30 and Cys16-Cys51. The structure of the inhibitor is homologous to Kazal-type trypsin inhibitors. The inhibitor constant, KI, for trypsin inhibition was 0.05 nM whereas chymotrypsin and elastase were not inhibited. To reveal the complete sequence the cDNA encoding the trypsin inhibitor was isolated. This cDNA of 454 bp predicts a protein of 82 amino acid residues including a 20 amino acid signal peptide. Moreover, the cDNA predicts a C-terminal extension of 11 amino acids compared to the part identified by protein sequencing. The molecular mass calculated for this predicted protein is in accordance with the measured value. This C-terminal sequence is unusual for Kazal-type trypsin inhibitors and has apparently been lost early in evolution. The high degree of conservation around the active site strongly supports the importance of the Kazal-type inhibitors.
Odum
L
L
Department of Clinical Biochemistry, Roskilde Hospital, Denmark.
Bundgaard
J R
JR
Johnsen
A H
AH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002938:Ciona intestinalis / Q000737:chemistry*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004220:Disulfides / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013820:Thermolysin / Q000378:metabolism; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000737:chemistry* / Q000235:genetics
D004220:Disulfides; D014359:Trypsin Inhibitor, Kazal Pancreatic; D013820:Thermolysin