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Isolation and partial characterization of a protein with HMG-CoA reductase phosphatase activity associated with rat
G Asins1, D Serra, F G Hegardt
1Unit of Biochemistry, University of Barcelona, School of Pharmacy, Spain.
Abstract:
Several rat liver HMG-CoA-reductase (HMG-CoA-Rd) phosphatase activities have been shown to be associated with the endoplasmic reticulum. These activities were not due to glycogen contamination, as judged not only from different patterns of solubilization of the microsomal membranes and the glycogen pellet but also by differential centrifugation behavior under standard conditions and in a sucrose gradient. We present evidence that at least three forms of protein phosphatase are associated with microsomal membranes: a polycation-stimulated type 2A phosphatase, a type 2C phosphatase, and a non-2A, non-2B, non-2C phosphatase. This last HMG-CoA-Rd phosphatase activity corresponding to an 85 kDa protein was partially purified by several chromatographic procedures. The IC50 value for the inhibition of the HMG-CoA-Rd phosphatase by I-2 was 10-fold higher than for the inhibition of the purified type 1 catalytic subunit from rabbit skeletal muscle. The microsomal HMG-CoA-Rd phosphatase activity was slightly affected by the protein inhibitor that inhibits type 2A activity when HMG-CoA reductase is the substrate. The HMG-CoA-Rd phosphatase activity is spontaneously active and it is not reactivated in the presence of Mg2+ or polycations. The holoenzyme does not contain the inhibitor-2 and it is not reactivated by incubation with ATP and glycogen synthase kinase-3. Proteolytic treatment of the enzyme yielded a polypeptide fragment of low Mr (37 kDa) with reduced activity. A model of holoenzymatic HMG-CoA-Rd phosphatase and its relation to the microsomal membranes is presented.
Insights
Researchers identified three protein phosphatase activities linked to endoplasmic reticulum membranes, including a novel HMG-CoA reductase phosphatase. This enzyme is spontaneously active and distinct from known phosphatases, offering new insights into cholesterol metabolism regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- HMG-CoA reductase (HMG-CoA-Rd) phosphatase activities are primarily associated with the endoplasmic reticulum in rat liver.
- Previous studies suggested these activities might be artifacts of glycogen contamination, but this has been contested.
- Understanding these phosphatases is crucial for regulating cholesterol synthesis.
Purpose of the Study:
- To characterize the HMG-CoA reductase phosphatase activities associated with rat liver microsomal membranes.
- To differentiate these activities from known phosphatase types and glycogen contamination.
- To elucidate the properties and potential structure of a novel HMG-CoA reductase phosphatase.
Main Methods:
- Differential centrifugation and sucrose gradient analysis to separate microsomal membranes and glycogen pellets.
- Solubilization studies to assess enzyme association with membranes.
- Chromatographic purification of the 85 kDa HMG-CoA-Rd phosphatase.
- Enzyme inhibition assays using specific inhibitors (I-2, protein inhibitor) and activators (Mg2+, polycations, ATP, glycogen synthase kinase-3).
- Proteolytic treatment to analyze enzyme subunits.
Main Results:
- Evidence for at least three distinct protein phosphatase activities in microsomal membranes: a type 2A, a type 2C, and a novel non-2A/2B/2C phosphatase.
- The novel HMG-CoA-Rd phosphatase, an 85 kDa protein, was partially purified and showed distinct properties.
- This phosphatase was spontaneously active, not requiring Mg2+ or polycation reactivation, and did not contain inhibitor-2.
- Its inhibition by I-2 was 10-fold less potent than for type 1 phosphatase, and it was only slightly affected by a type 2A inhibitor.
- Proteolytic cleavage yielded a 37 kDa fragment with reduced activity.
Conclusions:
- Rat liver endoplasmic reticulum harbors multiple HMG-CoA reductase phosphatase activities, including a novel, spontaneously active form.
- This novel phosphatase is distinct from known types 1, 2A, and 2C phosphatases and is not regulated by common reactivation mechanisms.
- The findings suggest a unique holoenzymatic structure for this microsomal HMG-CoA-Rd phosphatase, potentially playing a specific role in cholesterol metabolism regulation.