Proteolytic activities in plasma membrane preparations from rat liver. 2. Partial purification and characterization of membrane bound endopeptidases, dipeptidyl-aminopeptidase IV and aminopeptidase.
Plasma membranes (PM) were prepared from nuclear and microsomal fractions of rat liver as described in the preceding paper. Four different proteolytic activities were studied and found to be solubilized from PM fractions after detergent treatment: endopeptidase activity at neutral and acid pH, dipeptidyl-aminopeptidase IV, and (alanine-)aminopeptidase. Both PM preparations contain a serine-endopeptidase with optimal activity against azocasein at pH 7.6. After solubilization of PM derived from microsomal fractions (PM-m) and gel filtration this activity shows an apparent molecular mass of 220 +/- 20 kD. This membrane proteinase is different from other known serine proteinases of liver cells because of its large molecular mass and an activating effect of 1,10-phenanthroline. PM derived from microsomal fractions contain additional endopeptidase with a pH optimum of 5.2 that could be inhibited by 4-chloromercuribenzoate, leupeptin and Z-Phe-Ala-diazomethylketone. Using detergent solubilization and gel filtration this acid endopeptidase activity in PM-m can be resolved into three peaks with apparent molecular masses (detergent forms) of 180 +/- 10, 80 +/- 10 and 35 +/- 10 kD, the latter may be cathepsin L. In addition to the endopeptidases, PM-m were shown to contain also aminopeptidases degrading Ala-Pro-pNA and Ala-pNA. The former activity, dipeptidyl-aminopeptidase IV (DPP IV), has features similar to DPP IV from other microvillus membranes (inhibition by DIFP and PMSF, pH optimum at 7.7). The Ala-pNA degrading aminopeptidase is inhibited by chelating agents and some bivalent heavy metal ions, but is activated by Co++-ions. Both enzymes apparently were eluted as monomers (molecular mass 180-190 kD) after gel filtration in detergent containing buffers.
D000626:Aminopeptidases / Q000032:analysis*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000302:isolation & purification*; D010450:Endopeptidases / Q000032:analysis* / Q000302:isolation & purification*; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D051381:Rats; D013552:Swine
D010450:Endopeptidases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases