Disulfide bridge structure of ascidian trypsin inhibitor I: similarity to Kazal-type inhibitors.
The primary structures of ascidian trypsin inhibitors (iso-inhibitors I and II) were reported in the preceding paper (Kumazaki, T. et al. (1990) J. Biochem. 107, 409-413). Both of them have eight half-cystines in a molecule composed of 55 amino acid residues with a sequence showing no extensive homology to other known protease inhibitors. To locate the four disulfide bridges in the molecule, native inhibitor I was digested with thermolysin to yield cystine-containing peptides. The peptides were separated from each other by reversed-phase HPLC. A core peptide still containing six closely located half-cystines (e.g. -Cys-Arg-Cys and -Cys-Cys-) was further digested with Streptomyces griseus trypsin for cleavage of the Arg-Cys bond. On the other hand, the Cys-Cys bond was split by applying manual Edman degradation to the core peptide. Amino acid composition analyses of the resulting cystine peptides allowed us to define the whole disulfide bridge structure in the parent molecule. The topological relation between the disulfide loops and the reactive site suggested that the ascidian trypsin inhibitor may be classified as a member of the Kazal-type inhibitor family.
Kumazaki
T
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University.
Ishii
S
S
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003553:Cystine / Q000032:analysis; D004220:Disulfides / Q000032:analysis; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis; D013305:Streptomyces griseus / Q000201:enzymology; D013820:Thermolysin; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000032:analysis*; D014361:Trypsin Inhibitors / Q000032:analysis*; D014561:Urochordata / Q000378:metabolism*
D004220:Disulfides; D010455:Peptides; D014361:Trypsin Inhibitors; D003553:Cystine; D014359:Trypsin Inhibitor, Kazal Pancreatic; D013820:Thermolysin