Related Experiment Videos
Phosphorylation of microsomal HMG CoA reductase increases susceptibility to proteolytic degradation in vitro
Abstract:
Conversion of native, 97-100 kDa rat liver microsomal HMG CoA reductase to membrane-bound 62 kDa and soluble 52-56 kDa catalytically active forms was catalyzed in vitro by the calcium-dependent, leupeptin- and calpastatin-sensitive protease calpain-II purified from rat liver cytosol. Cleavage of the native 97-100 kDa reductase was enhanced by pretreatment (inactivation) of microsomes with ATP(Mg2+) and liver reductase kinase (compared to protein phosphatase-pretreated controls). This was reflected in a loss of the 97-100 kDa species and an increase in the soluble 52-56 kDa species (total enzyme activity and specific immunoblot recovery).
Insights
Rat liver calpain-II protease converts native HMG CoA reductase into smaller, active forms. Pretreatment with ATP and liver reductase kinase enhances this conversion, increasing soluble enzyme activity.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- HMG CoA reductase is a key enzyme in cholesterol biosynthesis.
- The enzyme exists in different forms, including native microsomal and soluble active forms.
- Proteolytic cleavage is implicated in regulating enzyme activity.
Purpose of the Study:
- To investigate the in vitro conversion of native rat liver HMG CoA reductase.
- To identify the protease responsible for cleaving HMG CoA reductase.
- To determine the effect of enzyme modification on cleavage.
Main Methods:
- Purification of calpain-II protease from rat liver cytosol.
- In vitro enzymatic assays using purified HMG CoA reductase and calpain-II.
- Microsomal pretreatment with ATP(Mg2+) and liver reductase kinase or protein phosphatase.
- Analysis of enzyme forms by SDS-PAGE and immunoblotting.
Main Results:
- Calpain-II catalyzed the conversion of native 97-100 kDa HMG CoA reductase to membrane-bound 62 kDa and soluble 52-56 kDa forms.
- Pretreatment of microsomes with ATP(Mg2+) and liver reductase kinase enhanced reductase cleavage.
- Enhanced cleavage resulted in decreased 97-100 kDa species and increased soluble 52-56 kDa species.
Conclusions:
- Calpain-II is a key protease involved in the conversion of HMG CoA reductase.
- Enzyme inactivation via phosphorylation enhances proteolytic cleavage by calpain-II.
- This process generates catalytically active soluble forms of HMG CoA reductase.