Intestinal peptide hydrolases differences between brush border and cytoplasmic enzymes

Abstract

Enzymes catalyzing hydrolysis of L-phenylalanylglycine (Phe-Gly) and glycyl- L-phenylalanine (Gly-Phe) were demonstrated in both cytoplasmic and brush border fractions of rat intestinal mucosa. The particulate fractions of mucosa comprised 61% of Phe-Gly hydrolase but only 3% of the total Gly-Phe hydrolase activity. In the presence of 0.1 mM p-hydroxymercuribenzoate (PHMB), both cytoplasmic activities were inhibited more than 95% but brush border hydrolases were unaffected. Heating for 1 h at 55 °C destroyed more than 95% of the soluble dipeptide hydrolase activities but decreased brush border activities less than 15%. In addition, Michaelis-Menten constants for both brush border enzymes were significantly greater than for the corresponding cytoplasmic activities. Brush border and supernatant enzymes had similar pH optima. Brush border hydirolases solubilized with Triton X-100, like the enzymes of intact bruch borders, were heat stable and not inhibited by PHMB. Assays of isolated epithelial cells confirmed the presence of dipeptide hydrolase activity in the cytoplasm of the enterocyte. Following ligation of the common pancreatico-biliary duct, neither cytoplasmic nor particulate peptide hydrolase decreased sufficiently to suggest that pancreatic enzymes contribute significantly to mucosal Phe-Gly or Gly-Phe hydrolase activities. The present observations strongly suggest that different enzymes are involved in the hydrolysis of dipeptides by brush border and cytoplasmic fractions of intestinal epithelial cells. It remains to be determined whether brush border or cytoplasmic enzymes, or both, are responsible for hydrolysis of peptides derived from intraluminal proteins.