Isolation of procathepsin D from mature cathepsin D by pepstatin affinity chromatography. Autocatalytic proteolysis of the zymogen form of the enzyme

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Abstract

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°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°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.

Original languageEnglish
Pages (from-to)601-604
Number of pages4
JournalBiochemical Journal
Volume263
Issue number2
StatePublished - Jan 1 1989

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Proteolysis
Affinity chromatography
Cathepsin D
Enzyme Precursors
Affinity Chromatography
Enzymes
Buffers
procathepsin D
pepstatin
Peptide Hydrolases
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Isolation of procathepsin D from mature cathepsin D by pepstatin affinity chromatography. Autocatalytic proteolysis of the zymogen form of the enzyme",
abstract = "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°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°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.",
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N2 - 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°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°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.

AB - 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°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°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.

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