Rabbit intestinal aminopeptidase N. Purification and molecular properties.
The detergent and protease forms of rabbit intestinal aminopeptidase N were purfied for chemical investigations and future specific immunological labeling of the enzyme in situ. The purification of the detergent form required a special technique called 'reverse immunoabsorbant chromatography'. The specific activity of the detergent form finally obtained was identical to that of the protease form. A significant charge micro heterogeneity persisted in the most purified preparations, due probably to a certain level of variability in the sugar moiety. The major proteolytic cleavage which occurred at the hydrophilic-hydrophobic junction of the detergent form during its conversion into the protease form was well defined. But additional splittings probably in C-terminal region of the molecules led to several protease forms differing by their size. The molecular weight assigned to the peptide liberated during the above conversion was overestimated due to preferential detergent binding to hydrophobic structures. The correct value, estimated by a new isotopic dilution method, was 3800 (36-38 residues) for the peptide originating from the rabbit enzyme. The real anchor plunging into the membrane core is possibly still shorter. Comparative N-terminal residue determinations in the detergent form, the protease form and the peptide difinitely confirmed that the enzyme is anchored to the bursh border membrane by its N-terminal region.
D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000302:isolation & purification*; D000818:Animals; D018826:CD13 Antigens; D002241:Carbohydrates / Q000032:analysis; D005779:Immunodiffusion; D007123:Immunoelectrophoresis, Two-Dimensional; D007413:Intestinal Mucosa / Q000201:enzymology*; D007422:Intestines / Q000201:enzymology*; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D014357:Trypsin
D000596:Amino Acids; D002241:Carbohydrates; D010446:Peptide Fragments; D000626:Aminopeptidases; D018826:CD13 Antigens; D014357:Trypsin