Selective Assay Method with Use of Heat Stability for a Trypsin-sensitive Cholecystokinin (CCK)-releasing Peptide (Monitor Peptide) in Rat Bile-pancreatic Juice

Abstract

A simple and selective assay method for a trypsin-sensitive cholecystokinin (CCK)-releasing peptide (‘monitor peptide’) found in rät bile-pancreatic juice is reported. This peptide also has trypsin inhibitory activity which could be used as an index for measurement, except that it was difficult to assay the inhibitory activity of the peptide directly in the bile-pancreatic juice because of abundant endogenous trypsin. The peptide and trypsin were purified from rat bile-pancreatic juice, and their sensitivities to heat treatment was examined. Trypsin(ogen) was completely eliminated by being heated at 80°C for 40min in the presence of 0.5 m NaCl, pH 2.5, whereas the trypsin inhibitory activity of the peptide was above 95% of the original level after this treatment. Using the difference, we established assay conditions for trypsin inhibition by the peptide in which the effects of endogenous trypsin were removed by heating. This assay method was then used to show that there was a close relationship between trypsin(ogen) and the peptide in the bile-pancreatic juice. We estimated that the trypsin inhibitory activity of the peptide masks 2–5% of the trypsin activity in the bile-pancreatic juice from Wistar male rats fed a stock diet. The optimum conditions for activation of trypsinogen and chymotrypsinogen in rat bile-pancreatic juice were also studied. At 37°C, trypsinogen and chymotrypsinogen were maximally activated by enterokinase 5% of protein being activated for 40 and 6 min, respectively.