Journal of Lipid Research, Vol 32, 1391-1401, Copyright © 1991 by Lipid Research, Inc.
Isolation and partial characterization of a protein with HMG-CoA reductase phosphatase activity associated with rat liver microsomal membranes
G Asins, D Serra and FG Hegardt
Unit of Biochemistry, University of Barcelona, School of Pharmacy, Spain.
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.
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