Stabilization of the C-terminal part of pig and horse colipase by carboxypeptidase and trypsin inhibitors.

The research article discusses the stabilization of pig and horse colipases using trypsin and carboxypeptidase inhibitors. The researchers identified two forms of pig colipase and four forms of horse colipase, which are more susceptible to proteolytic degradation at their C-terminal parts than their N-terminal parts.

Research Method

• The researchers purified pig and horse colipases using a common procedure that involved trypsin and carboxypeptidase inhibitors as stabilizers. They studied the effect of these inhibitors on the degradation process of the colipases.

Pig Colipase

• There were two forms of pig colipase identified. The predominant A1 form contained about 103-105 residues.
• The minor and slightly degraded A2 form lacked the last two C-terminal residues, Asp and Ser.
• Carboxypeptidase inhibitors were found to significantly slow down the degradation of this variant.

Horse Colipase

• The research identified a total of four forms of the horse cofactor.
• The two primary forms, A1 and B1, were likely isocolipases that differed by only a few substitutions and contained the same number of residues (about 96).
• The A2 and B2 variants are believed to be degraded forms probably due to tryptic cleavage of the Arg-Ser bond in the sequence.
• They featured an N-terminal valine and an Arg-Ser-Glu-(Glx)1,2-ArgC-terminal sequence.
• Due to the presence of methionine in the horse cofactor, fragmentation was possible with cyanogen bromide, allowing further investigation into its structure.

Results and Conclusion

• The authors concluded that the C-terminal parts of both pig and horse colipases are more susceptible to proteolytic degradation than their N-terminal counterparts.
• Implying that the inhibitors used, provide more stability to these exposed regions, thereby preventing fast degradation.
• Preliminary steps to crystallize one of the variants of the horse colipase (B1) were also taken to gain more insight into its structure and properties.