1. Proteolytic activities in plasma membrane preparations from rat liver. 2. Partial purification and characterization of membrane bound endopeptidases, dipeptidyl-aminopeptidase IV and aminopeptidase
E Schön Biomed Biochim Acta. 1983;42(5):451-64.
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.
2. Recombinant expression and purification of an enzymatically active cysteine proteinase of the protozoan parasite Entamoeba histolytica
A Hellberg, N Nowak, M Leippe, E Tannich, I Bruchhaus Protein Expr Purif. 2002 Feb;24(1):131-7. doi: 10.1006/prep.2001.1548.
Cysteine proteinases and in particular cysteine proteinase 5 (EhCP5) of Entamoeba histolytica are considered important for ameba pathogenicity. To study EhCP5 in more detail a protocol was elaborated to produce considerable amounts of the enzyme in its active form. The protein was expressed in Escherichia coli as a histidine-tagged pro-enzyme and purified to homogeneity under denaturing conditions in the presence of guanidine-HCl using nickel affinity chromatography. Renaturation was performed by 100-fold dilution in a buffer containing reduced and oxidized thiols, which led to soluble but enzymatically inactive pro-enzyme. Further processing and activation was achieved in the presence of 10 mM DTT and 0.04% SDS at 37 degrees C. Recombinant enzyme (rEhCP5) was indistinguishable from native EhCP5 purified from E. histolytica lysates. Both runs in SDS-PAGE under reducing and nonreducing conditions at positions corresponding to 27 and 29 kDa, respectively, had the same pH optima and displayed similar specific activity against azocasein. Moreover, both enzymes were active against a broad spectrum of biological and synthetic substrates such as mucin, fibrinogen, collagen, human hemoglobin, bovine serum albumin, gelatin, human IgG, Z-Arg-Arg-pNA, and Z-Ala-Arg-Arg-pNA, but not against Z-Phe-Arg-pNA. The identity of rEhCP5 as a cysteine proteinase was confirmed by inhibition with specific cysteine proteinase inhibitors. In contrast, various compounds known to specifically inhibit aspartic, metallo, or serine proteinases had no effect on rEhCP5 activity.
