Purification and Characterization of an Aminopeptidase A from Hog Intestinal Brush‐Border Membrane

The aminopeptidase A of the porcine intestinal brush‐border membrane has been purified following solubilization by trypsin (p‐form) or Emulphogen (d‐form). Full purification of d‐amino‐peptidase A required the use of anti‐impurities immunoabsorbant chromatography. The d‐amino‐peptidase A constitutes about 4% of the total proteins of the membrane, compared to 8–12% for another, already characterized, brush‐border aminopeptidase N.Both d‐form and p‐form of aminopeptidase A have been clearly shown to be dimeric. Experimental evidence is presented favoring the view that they are symmetrical dimers, with the consequence that each of the two subunits of the d‐form possesses an hydrophobic anchor holding them at the membrane surface. As already demonstrated for several other brush border hydrolases, the hydro‐phobic anchor is N‐terminal in porcine intestinal aminopeptidase A.The molecular weight of the peptide including the anchor liberated by trypsin during the conversion of the d‐form into the p‐form has been estimated by an isotopic dilution method to be about 4500 (42 residues). This value which compares well with those recently obtained in the case of rabbit aminopeptidase N (3700–3800; 36–38 residues), indicates that the anchor is much shorter than believed earlier.A preliminary survey of the specificity of both aminopeptidases A and N towards four synthetic amino acid p‐nitroanilides confirms that aminopeptidase A mostly cleaves acidic residues. Its activity towards neutral residues is much lower, but probably significant in certain cases.