Purification of Different Amphiphilic Forms of a Microvillus Aminopeptidase from Pig Small Intestine Using Immunoadsorbent Chromatography

Abstract

1 A detergent-solubilized pig intestinal microvillus aminopeptidase (microsomal aminopeptidase, EC 3.4.11.2) was purified approximately 150 times with a yield of about 20% by a rapid and simple immunoadsorbent technique. The purified enzyme was shown to be homogeneous by crossed immunoelectrophoresis and by polyacrylamide gel electrophoresis in dodecyl sulphate. Furthermore it was essentially free from other known peptidases and disaccharidases. 2 By charge-shift crossed immunoelectrophoresis it was demonstrated that the hydrophilic form was continuously generated both during a more time-consuming purification procedure and during storage. The development of a rapid purification procedure allowed the preparation of an amphiphilic form free from the hydrophilic one, which could be stabilized by 0.5 mM HgCl2. 3 Intestinal aminopeptidase isolated from a pig having the pancreatic duct ligated three days before sacrifice migrated in polyacrylamide gel electrophoresis in dodecyl sulphate as a single polypeptide chain with a molecular weight of 162000. In contrast, the enzyme from a pig with intact pancreatic duct when purified even in the presence of Kunitz trypsin inhibitor consisted of three polypeptide chains (molecular weights 162000, 123000 and 61000). Trypsin treatment in vitro converted the large polypeptide chain into the two smaller ones. It is suggested that the membrane-bound enzyme after biosynthesis is modified by trypsin and/or chymotrypsin. 4 The conversion of the amphiphilic form to the hydrophilic one and the hydrolysis of the large polypeptide chain into the two smaller ones represented different reactions.