Crayfish carboxypeptidase. Affinity chromatography, characterization and amino-terminal sequence

Area of Science:

• Biochemistry
• Evolutionary Biology
• Enzymology

Background:

• Pancreatic metalloexopeptidases play crucial roles in digestion.
• Understanding their evolutionary history requires comparative studies across species.
• Crayfish represent a divergent lineage for evolutionary insights.

Purpose of the Study:

• To isolate and characterize carboxypeptidase from the crayfish Astacus fluviatilis cardia.
• To investigate the evolutionary relationship between crayfish and mammalian pancreatic carboxypeptidases.

Main Methods:

• Enzyme isolation using affinity chromatography with potato carboxypeptidase inhibitor.
• Characterization of enzyme specificity, pH optimum, and isoelectric point.
• Determination of metal cofactor (zinc) via metal chelating agents and neutron activation analysis.
• Analysis of amino acid composition and N-terminal sequencing.

Main Results:

• Successfully isolated pure crayfish carboxypeptidase, yielding 25 mg from 50 ml cardia fluid.
• The enzyme exhibits specificity similar to bovine carboxypeptidase B.
• Confirmed crayfish carboxypeptidase as a zinc metalloenzyme.
• Amino acid composition shows similarity to mammalian counterparts, with higher acidic residue content.
• N-terminal sequencing revealed significant homology to pancreatic carboxypeptidases A and B.

Conclusions:

• Crayfish carboxypeptidase is a zinc metalloenzyme with functional and structural similarities to mammalian pancreatic carboxypeptidases.
• These findings support an evolutionary link between invertebrate and vertebrate carboxypeptidases.
• The study provides insights into the evolution of digestive enzymes.