Endopeptidase of the Brush Border Membrane of Rat Enterocyte. Separation from Aminopeptidase and Partial Characterization

Abstract

The brush border of rat enterocytes was isolated by differential centrifugation with CaCl2 according to Schmitz. This material was solubilized with papain (EC 3.4.22.2), trypsin (EC 3.4.21.4) and Triton X-100. The greatest amount of membrane enzymes was released in the supernatant (105,000 .times. g) with the use of Triton X-100. The tritonized supernatant was then treated with papain, bromelain (EC 3.4.22.4), ficin (EC 3.4.22.3) and trypsin (individually or in combinations). After simultaneous proteolysis with papain and bromelain a partial separation of the aminopeptidase from the endopeptidase by Sephadex G-200 chromatography was observed. These 2 enzyme activities were distinctly separated by isoelectric focusing at pH 4-6. Two enzymatically active bands (Rf 0.13 and 0.24) in the aminopeptidase fraction and 1 single active band (Rf 0.16) in the endopeptidase fraction using polyacrylamide gel electrophoresis were found. Co-migrating proteins to all of these activities were detected.