Isolation of procathepsin D from mature cathepsin D by pepstatin affinity chromatography. Autocatalytic proteolysis of the zymogen form of the enzyme.
Procathepsin D is a rapidly processed precursor form of the lysosomal proteinase cathepsin D. The enzymic properties of procathepsin D have been studied by examining the pepstatin-binding characteristics of both the precursor and the mature enzyme. Procathepsin D bound to immobilized pepstatin at 4 degrees C in pH 3.5 buffer but not in pH 5.3 buffer, whereas mature forms of cathepsin D bound to immobilized pepstatin at both pH values. These characteristics of procathepsin D were exploited to isolate the proenzyme from mature forms and to determine whether activation of the proenzyme is an autocatalytic process. After incubation at 37 degrees C in pH 3.5 buffer, the proenzyme underwent pepstatin-inhibitable proteolysis resulting in a dramatically increased affinity of purified procathepsin D for pepstatin at pH 5.3. The low concentration of enzyme used in these studies suggests that procathepsin D cleavage to single-chain cathepsin D may occur via a unimolecular mechanism.
Conner
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Department of Cell Biology and Anatomy, University of Miami School of Medicine, FL 33101.
D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002402:Cathepsin D / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009842:Oligopeptides / Q000378:metabolism*; D010436:Pepstatins / Q000378:metabolism*; D013552:Swine
D004792:Enzyme Precursors; D009842:Oligopeptides; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; C058345:procathepsin D; D002402:Cathepsin D; C031375:pepstatin