Purification and characterization of a protein inhibitor from rat

A protein inhibitor of HMG-CoA reductase phosphatase activity from rat liver was purified to homogeneity. The protein was purified 4,000-fold with an overall yield of 4%. The purified protein had a molecular mass of 31 kDa. This spontaneously active protein is thermostable and acid-resistant. The protein inhibitor is phosphorylated by glycogen synthase kinase-3 and CAMP-dependent protein kinase without change in its inhibitory activity. The inhibition caused by this inhibitor on phosphatases 1 and 2A is similar to that of inhibitor-2 from rabbit skeletal muscle using hydroxymethylglutaryl-CoA reductase as substrate. The regulation properties of this inhibitor towards phosphatase 1 together with another protein inhibitor of phosphatase 2A in cholesterol metabolism are discussed. Serra, D., G. Aeins, and F. G. Hegardt. Purification and characterization of a protein inhibitor from rat liver that inhibits type 1 protein phosphatase when 3-hydroxy-3-methylglutaryl CoA reductase is the substrate. J. Lipid Res. 1990. 31: 919-926. Supplementary key words HMG-CoA reductase phosphatases protein phosphorylation inhibitor-2 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMGCoA) reductase (E.C. 1.1.1.34) the main rate-limiting enzyme for synthesis of cholesterol and isoprenoids, has been shown to be regulated by covalent modification. Reductase kinase inactivates and phosphorylates HMG-CoA reductase both in vitro and in vivo, this process being activated by AMP (1, 2). Inactivated, homogeneous 32P-labeled HMG-CoA reductase is reactivated in vitro by rat liver type 1 and type 2 protein phosphatases with a concomitant release of 32P (3). Type 1 protein phosphatases are inhibited at nanomolar concentration by the heat-stable proteins, termed inhibitor-1 and inhibitor-2 (4-6). In a different way, type 2 protein phosphatases are inhibited at much higher concentrations by inhibitor-2 (7). Inhibitor-1 described by Huang and Glinsman (4) requires phosphorylation by the cyclic AMP-dependent protein kinase for its activity. In contrast, inhibitor-2 is active without phosphorylation and usually copurifies with the phosphatase 1 as an inactive complex with a molecular mass of 70 kDa (8, 9). Rabbit liver inhibitor-2 was purified to apparent homogeneity by Khandelwal and Zinman (lo), and had an apparent molecular mass of 15 kDa by SDS-PAGE. Later, Chisholm and Cohen (11) reported that the partially purified inhibitor-2 from rabbit liver had the same molecular mass (30.5 kDa) as the inhibitor-2 from rabbit skeletal muscle. There is no previous report on the purification to homogeneity of a rat liver protein inhibitor active on protein phosphatase 1. In this report, we describe a procedure for the purification of a protein inhibitor of phosphatase type 1 from rat liver using HMG-CoA reductase as the substrate. The purified protein was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of SDS. The molecular mass of the rat liver inhibitor determined by SDS-polyacrylamide gel electrophoresis was found to be 31 kDa. This protein inhibitor showed kinetic properties towards protein phosphatases 1 and 2A similar to rabbit muscle inhibitor-2 using as substrates not only HMG-CoA reductase but also phosphorylase. MATERIALS AND METHODS