Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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).
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PMID:Phosphorylation of microsomal HMG CoA reductase increases susceptibility to proteolytic degradation in vitro. 609 45

We have investigated the comparative biochemistry of in vitro regulation of HMG-CoA reductase (EC 1.1.1.34) in microsomal preparations from the livers of nine vertebrates. In all instances, reductase activity was rapidly and profoundly decreased by addition of MgATP. Reductase activities were restored to near or above initial levels after removal of MgATP and incubation with a crude, low molecular weight phosphatase preparation from rat liver cytosol. Restoration of reductase activity was inhibited both by NaF and by pyrophosphate, known inhibitors of phosphoprotein phosphatase activity. Liver cytosol of species other than the rat exhibits reductase phosphatase activity. The converter enzymes that catalyze modulation of MG-CoA reductase activity (reductase kinase and reductase phosphatase) thus appear to be ubiquitous in vertebrate liver. Interconversion in vitro of active and inactive forms of reductase probably is general for vertebrate liver also. The majority of the reductase present in vertebrate liver may be present in a catalytically inactive or latent form in vivo. Under the experimental conditions used, the fraction present in the active form is, for a given species, quite constant. Species to species, from 20-45% of the reductase appears to be present in the active form.
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PMID:Regulation of vertebrate liver HMG-CoA reductase via reversible modulation of its catalytic activity. 624 8

We have previously reported that the enzymic activity of rat liver-3-hydroxy-3-methyl-glutaryl-CoA reductase (NADPH) (HMG-CoA reductase) is modulated in vitro by a phosphorylation-dephosphorylation reaction sequence. The in vitro phosphorylation of HMG-CoA reductase was further studied by utilizing purified HMG-CoA reductase and reductase kinase. Analysis of 32P-labeled HMG-CoA reductase revealed 1 mol of phosphate per subunit. Purified [32P]HMG-CoA reductase could be dephosphorylated with phosphoprotein phosphatase. To demonstrate the in vivo phosphorylation, rats were injected with 32P and hepatic HMG-CoA reductase was isolated by immunoprecipitation and also by purification of the enzyme to homogeneity. Analysis of [32P]HMG-CoA reductase by sodium dodecyl sulfate gel electrophoresis revealed a single peak of radioactivity comigrating with HMG-CoA reductase. Administration of glucagon enhances the in vivo phosphorylation of both HMG-CoA reductase and reductase kinase. In response to glucagon, HMG-CoA reductase activity is decreased whereas reductase kinase activity is increased. These results support our concept that the enzymic activity of HMG-CoA reductase is modulated by a bicyclic cascade system involving phosphorylation-dephosphorylation. The enzymic activity of HMG-CoA reductase has also been shown to be modulated by cholesterol and mevalonolactone by both short-term and long-term mechanisms. The effects of cholesterol and mevalonolactone are twofold. Rapid inhibition of HMG-CoA reductase activity is due to increased phosphorylation of the enzyme; the long-term effect of HMG-CoA reductase is achieved by reduction in enzyme concentration by modulation of enzyme synthesis and/or degradation. Regulation of HMG-CoA reductase by mevalonolactone is of major importance in cellular metabolism because mevalonate serves as precursor for four separate metabolic pathways, including the formation of cholesterol, ubiquinone, dolichols, and isopentenyl tRNA.
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PMID:Modulation of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity by reversible phosphorylation. 628 63

Assay of HMG-CoA reductase kinase activity requires HMG-CoA reductase (reductase, E.C. 1.1.1.34) free of associated reductase kinase. Microsomal reductase insensitive to inactivation by Mg-nucleotides alone may be prepared by heating microsomes at 50 degrees C for 15 min. The reductase in these microsomes may subsequently be inactivated by Mg-nucleotides only after addition of reductase kinase. Inactivation is a linear function of time and of cytosol protein concentration and may be reversed by treatment with a phosphoprotein phosphatase. The extent of inactivation observed under standard conditions provides an assay for reductase kinase activity. Factors present in cytosol that hinder measurement of either reductase or reductase kinase activity must be removed or inhibited. Reductase phosphatase is inhibited by 50 mM NaF. Reductase kinase kinase activity is not expressed under the assay conditions used. Mg-Nucleotide-independent inhibitors of reductase activity are removed by chromatography on DEAE-Sephacel or Blue Sepharose. Mevalonate kinase and reductase kinase are separable by chromatography on DEAE-Sephacel or Sephadex G-200. We describe a rapid chromatographic procedure for separating reductase kinase of crude fractions from mevalonate kinase and from Mg-nucleotide-independent inhibitors of reductase activity. The 1.0 M KCl eluate from DEAE-Sephacel contains all of the cytosol reductase kinase activity. This method is applicable to measurement of reductase kinase activity in cytosol or more purified fractions.
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PMID:HMG-CoA reductase kinase: measurement of activity by methods that preclude interference by inhibitors of HMG-CoA reductase activity or by mevalonate kinase. 628 22

The protein phosphatase activities involved in regulating the major pathways of intermediary metabolism can be explained by only four enzymes which can be conveniently divided into two classes, type-1 and type-2. Type-1 protein phosphatases dephosphorylate the beta-subunit of phosphorylase kinase and are potently inhibited by two thermostable proteins termed inhibitor-1 and inhibitor-2, whereas type-2 protein phosphatases preferentially dephosphorylate the alpha-subunit of phosphorylase kinase and are insensitive to inhibitor-1 and inhibitor-2. The substrate specificities of the four enzymes, namely protein phosphatase-1 (type-1) and protein phosphatases 2A, 2B and 2C (type-2) have been investigated. Eight different protein kinases were used to phosphorylate 13 different substrate proteins on a minimum of 20 different serine and threonine residues. These substrates include proteins involved in the regulation of glycogen metabolism, glycolysis, fatty acid synthesis, cholesterol synthesis, protein synthesis and muscle contraction. The studies demonstrate that protein phosphatase-1 and protein phosphatase 2A have very broad substrate specificities. The major differences, apart from the site specificity for phosphorylase kinase, are the much higher myosin light chain phosphatase and ATP-citrate lyase phosphatase activities of protein phosphatase-2A. Protein phosphatase-2C (an Mg2+-dependent enzyme) also has a broad specificity, but can be distinguished from protein phosphatase-2A by its extremely low phosphorylase phosphatase and histone H1 phosphatase activities, and its slow dephosphorylation of sites (3a + 3b + 3c) on glycogen synthase relative to site-2 of glycogen synthase. It has extremely high hydroxymethylglutaryl-CoA (HMG-CoA) reductase phosphatase and HMG-CoA reductase kinase phosphatase activity. Protein phosphatase-2B (a Ca2+-calmodulin-dependent enzyme) is the most specific phosphatase and only dephosphorylated three of the substrates (the alpha-subunit of phosphorylase kinase, inhibitor-1 and myosin light chains) at a significant rate. It is specifically inhibited by the phenathiazine drug, trifluoperazine. Examination of the amino acid sequences around each phosphorylation site does not support the idea that protein phosphatase specificity is determined by the primary structure in the immediate vicinity of the phosphorylation site.
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PMID:The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities. 630 24

Methods were developed for quantifying protein phosphatases-1, 2A, 2B and 2C in cell extracts, and these procedures were exploited to determine their tissue and subcellular distributions. In addition, the contribution of each enzyme to the total protein phosphatase activity in skeletal muscle and liver extracts towards nine proteins involved in the control of glycogen metabolism, glycolysis/gluconeogenesis, fatty acid synthesis and cholesterol synthesis was assessed. Each protein phosphatase was present at significant concentrations in skeletal muscle, heart muscle, liver, brain and adipose tissue, although the relative amounts differed considerably. In skeletal muscle, protein phosphatase-1 was the major enzyme acting on phosphorylase, glycogen synthase and phosphorylase kinase (beta-subunit), and thus was the major protein phosphatase responsible for the inactivation of glycogenolysis and stimulation of glycogen synthesis. This idea was reinforced by the observation that 50% of the protein phosphatase-1 activity was associated with the protein-glycogen complex. In the liver, protein phosphatases-1, 2A and 2C each appear to play a role in the regulation of glycogen metabolism. Protein phosphatase-1 accounted for a significant fraction of the total potential activity towards phosphorylase and glycogen synthase, and was the major phosphorylase kinase (beta-subunit) phosphatase of this tissue. In addition, it was the only protein phosphatase present in the protein-glycogen complex. Protein phosphatase 2A was also a major phosphorylase phosphatase and glycogen synthase phosphatase in this tissue. Protein phosphatase 2C was a significant glycogen synthase phosphatase in the liver, but had negligible activity toward phosphorylase or phosphorylase kinase (beta-subunit). In the absence of Ca2+, protein phosphatase 2A was the major phosphorylase kinase (alpha-subunit) phosphatase and the only inhibitor-1 phosphatase, in skeletal muscle or liver. In the presence of Ca2+, protein phosphatase 2B accounted for most of the activity towards these substrates. Protein phosphatase 2A was the major enzyme acting on L-pyruvate kinase, ATP-citrate lyase and acetyl-CoA carboxylase in rat liver, suggesting an important role in the regulation of glycolysis/gluconeogenesis and fatty acid synthesis. Protein phosphatase 2C was the major enzyme acting on hydroxymethylglutaryl-CoA (HMG-CoA) reductase and HMG-CoA reductase kinase, suggesting an important role in the regulation of cholesterol synthesis. However, the observation that 20% of the protein phosphatase-1 in liver was associated with the microsomal fraction suggests that this enzyme may also be involved in regulating HMG-CoA reductase, which is tightly associated with microsomes. The activity of protein phosphatase-1 in dilute skeletal muscle and liver extracts was just as sensitive to inhibitor-1 and inhibitor-2 as the purified enzyme. In concentrated extracts, higher concentrations of the inhibitor proteins were required and the inhibition was time-dependent...
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PMID:The protein phosphatases involved in cellular regulation. 6. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. 630 29

A growing body of evidence indicates that 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34, reductase) is degraded by proteolytic enzymes during solubilization by traditional freeze-thaw techniques. We have solubilized reductase in an active, stable form with nonionic detergents [Lubrol WX or poly(oxyethylene) ether type W-1]. Solubilization proceeded in high (greater than 70%) yield in the presence of the proteolytic inhibitors leupeptin, phenylmethanesulfonyl fluoride, and ethylene glycol bis(beta-aminoethyl ether)-N,N,-N',N'-tetraacetic acid and was independent of prior freeze-thawing of the microsomes. We have purified detergent-solubilized reductase 40-fold in high yield by means of sucrose density gradient centrifugation and dye-ligand chromatography. Detergent-solubilized reductase is heat labile, unlike reductase solubilized by the freeze-thaw method. Detergent-solubilized reductase can be inactivated up to 90% by use of reductase kinase. This inactivation requires both adenosine 5'-triphosphate and adenosine 5'-diphosphate, as has been previously observed for both microsomal and freeze-thaw solubilized reductase. Inactivation is reversed by subsequent treatment with a phosphoprotein phosphatase.
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PMID:3-hydroxy-3-methylglutaryl-CoA reductase: solubilization in the presence of proteolytic inhibitors, partial purification, and reversible phosphorylation-dephosphorylation. 630 48

It has been previously demonstrated that the enzymic activity of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34) is modulated in vitro and in vivo by a bicyclic cascade system involving reversible phosphorylation of HMG-CoA reductase and reductase kinase. In the present study, administration of mevalonolactone to rats caused a rapid inhibition of HMG-CoA reductase activity. The initial short-term (20-min) reversible inhibition (38%) of enzyme activity was due to increased phosphorylation of HMG-CoA reductase. The inhibition of HMG-CoA reductase activity by increased phosphorylation was associated with an increased activity and phosphorylation (2- to 3-fold) of reductase kinase. The increased phosphorylation of reductase kinase was catalyzed by reductase kinase kinase, which was significantly elevated (3- to 4-fold) after the administration of mevalonolactone to rats. The mechanism for the in vivo activation of reductase kinase kinase is as yet unknown. Mevalonolactone administration was also associated with a significant inhibition of phosphoprotein phosphatase activity, which dephosphorylates both HMG-CoA reductase (activation) and reductase kinase (inactivation). These results indicate that mevalonolactone administration to rats in vivo was associated with an inhibition of HMG-CoA reductase activity by two mechanisms: (i) an increase in the degree of phosphorylation of both HMG-CoA reductase and reductase kinase due to increased activity of reductase kinase kinase; (ii) a decrease in the dephosphorylation of both HMG-CoA reductase and reductase kinase secondary to inhibition of phosphoprotein phosphatase activity. These combined effects favor an increase in the steady-state level of the phosphorylated forms of both HMG-CoA reductase and reductase kinase, resulting in a net reduction in the enzymic activity of HMG-CoA reductase and mevalonate formation. These results demonstrate that the activity of reductase kinase kinase is modulated in vivo, providing a mechanism for the regulation of the activities of both reductase kinase and HMG-CoA reductase.
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PMID:In vivo modulation of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase, reductase kinase, and reductase kinase kinase by mevalonolactone. 659 93

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.
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PMID:Short-term regulation of hydroxymethylglutaryl coenzyme A reductase by reversible phosphorylation: modulation of reductase phosphatase in rat hepatocytes. 705 70

Hormone sensitive lipase (HSL) is an enzyme of relatively broad specificity, having the ability to hydrolyze tri-, di- and mono-acylglycerols as well as cholesterol esters and small water-soluble substrates. This broad specificity allows HSL to perform a variety of functions in several tissues. A key feature of HSL is its ability to be activated via phosphorylation by cyclic AMP-dependent protein kinase. In addition it is phosphorylated at a second site by several kinases, notably AMP-activated protein kinase. Phosphorylation of this site apparently plays a role in rendering the enzyme hormone-insensitive, in that prior phosphorylation at site 2 prevents phosphorylation and activation at site 1 by cyclic AMP-dependent protein kinase. Investigation of the protein phosphatases responsible for dephosphorylation of these sites has indicated that phosphatase 2A plays a predominant role but also that protein phosphatase 2C is a significant phosphatase targeted against both phosphorylation sites. Evidence indicates that HSL has at least three functional domains which contain (a) the phosphorylation sites which control activity, (b) the active site responsible for the catalytic activity and (c) a lipid binding site responsible for anchoring the lipase at the water-lipid interface. Using limited proteolytic studies we have found that it is possible to cleave HSL into several fragments including a stable domain of M(r) approximately 17.6 kDa which contains the active site serine residue. Digestion under similar conditions also generates a stable domain of M(r) approximately 11.5 kDa containing both phosphorylation sites. Furthermore, under appropriate conditions it is possible to digest HSL and retain activity against water-soluble substrates but with the concomitant loss of activity against triacylglycerol, implying that a lipid binding domain is lost during this procedure. HSL is responsible for the neutral cholesterol esterase activity in macrophages and it may play a role in the accumulation of cholesterol esters which occur during the development of foam cells. HSL activity is reduced in macrophage foam cells, at least partly due to increased activity of a cytosolic HSL inhibitor protein. A finding unexplained for many years has been that, although lipolysis can be stimulated 50-100-fold in adipocytes by lipolytic hormones, HSL can apparently only be activated 2-3-fold via phosphorylation in vitro by cyclic AMP-dependent protein kinase. One possibility to explain this discrepancy is that an additional anchoring protein is missing from the in vitro system and indirect evidence is now accumulating for such a protein.
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PMID:The multifunctional role of hormone-sensitive lipase in lipid metabolism. 794 81


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