Rex A. Parker

Reversible modulation of the activities of both liver microsomal hydroxymethylglutaryl coenzyme a reductase and its inactivating enzyme. Evidence for regulation by phosphorylation-dephosphorylation

Abstract Rat liver microsomal hydroxymethylglutaryl CoA reductase is inactivated when microsomes are incubated with both ATP and Mg++ (1). Activity is fully restored with purified liver cytosolic phosphorylase phosphatase. The microsomal (Mg)ATP-dependent reductase inactivating enzyme (designated I) may be extracted and assayed in an I-deficient microsomal system. The soluble I preparation itself is inactivated with phosphorylase phosphatase. Inactive I can be reactivated in the presence of (Mg)ATP by an apparent cAMP-independent protein kinase in the microsomal extract. These findings are consistent with a model in which both hydroxymethylglutaryl CoA reductase and an associated protein kinase (I) are subject to reversible covalent modulation by phosphorylation-dephosphorylation.

Phosphorylation of microsomal HMG CoA reductase increases susceptibility to proteolytic degradation in vitro.

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).

Activation of the branched-chain α-ketoacid dehydrogenase complex by a broad specificity protein phosphatase

Summary A broad-specificity protein phosphatase, purified from rat liver, can be used to activate the phosphorylated (inactive) branched-chain α-ketoacid dehydrogenase complex of crude tissue extracts. This enables estimation of the proportion of active (unphosphorylated) complex in a given tissue under different physiological states. Practically all (95 percent) of the complex was found in the active form in rat hearts perfused with leucine as the only oxidizable substrate. In contrast, only 13 percent of the complex was found in the active form when the perfusion medium was supplemented with glucose plus insulin. These findings are consistent with previously measured flux rates through the complex in perfused rat hearts.

Short-term regulation of hydroxymethylglutaryl coenzyme A reductase by reversible phosphorylation: modulation of reductase phosphatase in rat hepatocytes.

Hydroxymethylglutaryl CoA reductase catalyzes the limiting step in cholesterol synthesis in liver and other tissues. Beginning in 1973 studies with subcellular systems established that microsomal reductase is inactivated with ATP(Mg) and reductase kinase, and restored to full activity with phospho-protein phosphatase. By contrast reductase kinase is inactivated with phosphatase and reactivated with a second protein kinase (reductase kinase kinase). This bicyclic system has now been confirmed in terms of homogeneous enzyme components and by direct reversible phosphorylation with [gamma 32P]ATP in several laboratories. Short-term endocrine control of reductase and reductase kinase has been demonstrated in intact rat hepatocytes. Preincubation of cells with glucagon brought about a fall in the expressed activity of reductase and a rise in reductase kinase consistent with net phosphorylation of both enzymes. Total reductase levels were also severely depressed after glucagon. Addition of insulin to suspensions of hepatocytes had the reverse effect on expressed activity of reductase (elevated) and reductase kinase (depressed). Insulin also prevented the decay in total reductase activity. Since both protein kinases identified in this system are cAMP-insensitive, it was possible that hormonal signaling is mediated through the protein phosphatase that acts on both reductase kinase and reductase. In recent studies we have shown that the rate of activation of endogenous reductase in hepatocyte extracts (microsomes plus cytosol) is responsive to hormonal modulation. Pretreatment of hepatocytes with insulin increases apparent reductase phosphatase activity in extracts while glucagon diminishes the rate of reductase activation. HMG CoA is converted to mevalonate by the reductase enzyme. In hepatocytes mevalonate is rapidly converted to cholesterol and to a variety of isoprene derivatives. Expressed reductase activity falls precipitously when hepatocytes are incubated with mevalonate (added in the form of mevalono-lactone). As in the case with glucagon pretreatment reductase phosphatase is rapidly diminished. (Mevalonate itself is not inhibitory to reductase or reductase phosphatase activity in subcellular systems.) It is probable that a product of mevalonate metabolism generated in intact cells may act as a reductase phosphatase inhibitor. Among these added inorganic pyrophosphate inhibited reductase phosphatase at low concentrations.

7 Hydroxymethylglutaryl-Coenzyme A Reductase

Publisher Summary This chapter discusses the findings that pertain principally to the control of hydroxymethylglutaryl-CoA (HMG-CoA) reductase through reversible phosphorylation. HMG-CoA reductase is an enzyme that is tightly bound to the endoplasmic reticulum of higher eucaryotic cells. HMG-CoA reductase appears to share the lipid environment of the endoplasmic reticulum with the terminal steroid-polyprenoid processing multienzyme systems, as well as with the lipid intermediates and products that are formed within the lipoprotein domain. Compactin, a fungal metabolite, binds and severely inhibits HMG-CoA reductase. Current information on the topology of the native HMG-CoA reductase enzyme places it among the intrinsic glycoproteins of the endoplasmic reticulum, securely anchored to the membrane with multiple hydrophobic regions attached to a catalytically active hydrophilic tail extending into the cytoplasm. This particular deployment probably permits the observed multivectorial regulation of HMG-CoA reductase and of cholesterol synthesis. It has been suggested that cholesterol or hydroxycholesterol may act as a repressor of HMG-CoA reductase synthesis, or may inhibit the activity of reductase directly in the endoplasmic reticulum by diminishing the fluidity of the lipoprotein structure in the immediate environment of the enzyme.

Synthesis and degradation of interconvertible enzymes. Kinetic equations of a model system

Steady-state and kinetic equations have been developed which characterize the rates of formation, interconversion, and degradation of an enzyme protein subject to reversible phosphorylation. The theoretical model system incorporates separate fractional degradative rate constants for the phosphorylated and dephosphorylated protein species. The classical models for interconvertible enzymes, and for protein turnover, are special limiting situations of the general model presented here.

Turnover of HMG CoA Reductase is Influenced by Phosphorylation

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (E.C. 1.1.1.34) in most metabolic contexts is the limiting step in cholesterol formation in mammalian liver and other tissues. The properties and significance of this enzyme have been extensively reviewed (Gibson 1985; Kennelly and Rodwell 1985).

REGULATION OF HMG-CoA REDUCTASE AND REDUCTASE KINASE BY REVERSIBLE PHOSPHORYLATION

Publisher Summary This chapter describes the regulation of HMG-CoA reductase and reductase kinase by reversible phosphorylation. Rat liver microsomal HMG-CoA reductase ( R ) is readily interconverted between an active (dephosphorylated) and an inactive (phosphorylated) state. Inactivation of R is catalyzed by R kinase ( RK ). This enzyme is present both in the cytosol and microsomes. Reactivation of R is catalyzed by a second cytosolic enzyme P which appears to be identical to the 35,000 dalton liver Phosphorylase phosphatase. RK was extracted from microsomes by repeatedly washing with neutral buffer. RK activity was assayed using R in RK -deficient microsomes as substrate. Substantial purification of soluble RK was achieved by chromatography on DEAE-cellulose. RK was found to have a molecular weight of 360,000 daltons by chromatography on Agarose A 0.5 m. Incubation of either soluble or microsomal RK at 37° resulted in a time-dependent inactivation of the enzyme. The rate of inactivation was greatly enhanced by purified P and inhibited by fluoride. RK was reactivated in a time-dependent manner by incubation with 2 mM MgATP in a low ionic strength medium. Reactivation was blocked in the high-ionic-strength medium routinely used to assay R and RK .