Intestinal surface amino-oligopeptidases. I. Isolation of two weight isomers and their subunits from rat brush border.

Aminopeptidases of the small intestinal brush border are strategically located to play a pivotal role in the assimilation of protein nutrients. but the membrane nature of these enzymes has made isolation difficult. Rat intestinal brush borders were solubilized by a papain .p-aminobenzyl cellulose complex and two aminopeptidases purified by zonal gradient centrifugation, Sephadex G-200 gel filtration, and polyacrylamide gel electrophoresis in a multiphasic buffer system with activity monitored by the specific substrate, Lleucyl+-naphthylamide. The final products, designated aminopeptidases I and II according to their molecular weights, were each homogeneous on polyacrylamide gel electrophoresis. Immunization of rabbits with aminopeptidase II produced a monospecific antiserum that yielded a single precipitin line of identity for aminopeptidases I and II in double immunodiffusion. Molecular weights of the enzymes and their subunits were determined in nondenaturing and sodium dodecyl sulfate (SDS)-acrylamide electrophoresis from the retardation coefficient determined from Ferguson plots. The molecular weight of aminopeptidase I was 320,000 -C 15.000 and that of aminopeptidase II was 180,000 f 20,000. These weight isomers were produced regardless of whether papain or Triton X-100 plus sonication was used for solubilization. Anomalously slow migration of subunits in SDS-acrylamide electrophoresis at a single total percentage of acrylamide produced falsely high molecular weights. When the molecular weights of the subunits were determined from the retardation coefficient based on three different values for total percentage acrylamide at constant per cent cross-linker, aminopeptidase II was found to contain a single subunit, Q, of 91,000 f 10,000 daltons and aminopeptidase I consisted of both the (Y subunit and a /I subunit (71,000 f 6,000 daltons). The data suggest that aminopeptidase II is a dimer of a subunits and aminopeptidase I is a tetramer made up of two (Y and two /3 subunits. Kinetic characteristics and the topography of the active site are considered in the accompanying paper.