Specificity of the Carboxypeptidase Inhibitor from Potatoes

Abstract

Carboxypeptidases from animal, plant, fungal, and bacterial sources were tested for their ability to bind to the carboxypeptidase inhibitor from Russet Burbank potatoes. Enzymes which participate in the degradation of dietary protein were partially purfied from animal species as diverse as the cow and the limpet, and all were potently affected by the inhibitor.However, several zymogens of the enzymes in this group were tested and shown not to bind immobiized inhibitor. With the exception of an enzyme from mast cells and a novel carboxypeptidase A-like enzyme from bovine placenta, all animal carboxypeptidases which were not of digestive tract origin were not affected by the inhibitor. The inhibitor had no effect on the enzymic activities of all plant and most microbial carboxypeptidases. However, a strong association between the inhibitor and Streptomyces griseus carboxypeptidase has been noted previously and a low afflnity (K, about 10 micromolar) for a carboxypeptidase G1 from an acinetobacterium was found in this study.Although proteinaceous inhibitors of the serine endopeptidases are widely distributed in the plant and animal kingdoms (for review see 18), only potatoes (26, 28), tomatoes (16), and roundworms (17) are known to contain specific inhibitors of the pancreatic carboxypeptidases. In this study we have attempted to identify which of a variety of carboxypeptidases are affected by the inhibitor from potatoes. This information is of interest in that it may elucidate the evolution of this enzyme class, determine which enzymes might be amenable to purification by affinity chromatography on immobilized inhibitor (1), and, perhaps, clarify the physiological role of the inhibitor. MATERIALS AND METHODSMaterials. Carboxypeptidase isoinhibitor II (CPI4) was purified from Russet Burbank potatoes as described by Ryan et al. (28,15). CPI was coupled to CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals) as described previously (I). Derivatized resin had a capacity of 6 mg of bovine carboxypeptidase A bound per ml of packed gel. All peptide and ester substrates were purchased from '

Morphologic, Phylogenetic and Chemical Characterization of a Brackish Colonial Picocyanobacterium (Coelosphaeriaceae) with Bioactive Properties

“…The carboxypeptidase A inhibition assays were modified from described methods [ 62 , 63 ]. Carboxypeptidase A from bovine pancreas (Sigma-Aldrich, St. Louis, MO, USA) was dissolved (50 µg·mL −1 ) in buffer solution (50 mM Tris-HCl, Sigma-Aldrich, St. Louis, MO, USA, pH 7.5).…”