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Identification of a precursor form of cathepsin D in microsomal lumen: characterization of enzymatic activation and
Y Nishimura1, M Higaki, K Kato
1Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Abstract:
A precursor form of cathepsin D with 45 kDa was demonstrated in the rat liver microsomal lumen by immunoblotting analysis. The microsomal fraction containing procathepsin D which passed through a pepstatin-Sepharose resin showed no appreciable activity of cathepsin D. The in vitro incubation of this fraction at pH 3.0 resulted in a gradual increase of proteolytic activity toward hemoglobin as substrate and also, the proteolytic conversion of procathepsin D to the mature form was concomitantly observed. The proteolytic processing step was sensitive to pepstatin. These results suggest that procathepsin D is inactive in the endoplasmic reticulum and may be converted to the active forms by autoproteolytic processing mechanism at acidic pH during biosynthesis.
Insights
Procathepsin D, an inactive precursor enzyme found in rat liver endoplasmic reticulum, is converted to its active form at acidic pH. This autoproteolytic processing is sensitive to pepstatin.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Cathepsin D is a key aspartic protease involved in protein degradation.
- The precursor form, procathepsin D, is synthesized in the endoplasmic reticulum.
- Understanding the activation mechanism of procathepsin D is crucial for elucidating its physiological roles.
Purpose of the Study:
- To investigate the activation mechanism of procathepsin D in rat liver microsomes.
- To determine the conditions under which procathepsin D is converted to its mature, active form.
Main Methods:
- Immunoblotting analysis was used to detect procathepsin D in rat liver microsomes.
- Pepstatin-Sepharose chromatography was employed to isolate procathepsin D.
- In vitro incubation at acidic pH (3.0) was performed to assess proteolytic activity and processing.
Main Results:
- A 45 kDa precursor form of cathepsin D (procathepsin D) was identified in the rat liver microsomal lumen.
- Microsomal fractions containing procathepsin D showed no inherent cathepsin D activity.
- Incubation at pH 3.0 led to increased proteolytic activity and conversion of procathepsin D to the mature form, a process inhibited by pepstatin.
Conclusions:
- Procathepsin D is enzymatically inactive within the endoplasmic reticulum.
- Acidic pH triggers autoproteolytic processing of procathepsin D to its active form during biosynthesis.
- Pepstatin effectively inhibits the activation of procathepsin D.