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 hydrophobic 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.
D000626:Aminopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D018826:CD13 Antigens; D002462:Cell Membrane / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D043384:Glutamyl Aminopeptidase; D007422:Intestines; D046911:Macromolecular Substances; D008871:Microvilli / Q000201:enzymology*; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D013552:Swine; D014461:Ultracentrifugation
D003902:Detergents; D046911:Macromolecular Substances; D010455:Peptides; D000626:Aminopeptidases; D018826:CD13 Antigens; D043384:Glutamyl Aminopeptidase