Isolation and partial characterization of a protein with HMG-CoA reductase phosphatase activity associated with rat liver microsomal membranes (original) (raw)
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The Journal of cell biology, 1979
A detailed investigation was conducted to determine the precise subcellular localization of the rate-limiting enzymes of hepatic glycogen metabolism (glycogen synthase and phosphorylase) and their regulatory enzymes (synthase phosphatase and phosphorylase phosphatase). Rat liver was homogenized and fractionated to produce soluble, rough and smooth microsomal fractions. Enzyme assays of the fractions were performed, and the results showed that glycogen synthase and phosphorylase were located in the soluble fraction of the livers. Synthase phosphatase and phosphorylase phosphatase activities were also present in soluble fractions, but were clearly identified in both rough and smooth microsomal fractions. It is suggested that the location of smooth endoplasmic reticulum (SER) within the cytosome forms a microenvironment within hepatocytes that establishes conditions necessary for glycogen synthesis (and degradation). Thus the location of SER in the cell determines regions of the hepato...
Phosphorylation of microsomal HMG CoA reductase increases susceptibility to proteolytic degradation
Biochemical and Biophysical Research Communications, 1984
Conversion of native, 97-100 k&t rat liver micmsoml HHG CoA reductase to mmbrane-bound 62 k&a and soluble 52-56 kDa catalytically active forms was catalyzed g vitro by the calcium-dependent, leupeptin-and calpastatin-sensitive protease calpam--purified from rat liver cytosol. Cleavage of the native 97-lOq+kDa xeductase was enhanced by pretreatment (inactivation) of micmsoms with ATP(Mg ) 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 kI& species (total enzyme activity and specific imnunoblot recovery).
The EMBO journal, 1990
The intact, 100 kd microsomal enzyme and the 53 kd catalytic fragment of rat HMG-CoA reductase are both phosphorylated and inactivated by the AMP-activated protein kinase. Using the catalytic fragment, we have purified and sequenced peptides containing the single site of phosphorylation. Comparison with the amino acid sequence predicted from the cDNAs encoding other mammalian HMG-CoA reductases identifies this site as a serine residue close to the C-terminus (Ser872 in the human enzyme). Phosphopeptide mapping of native, 100 kd microsomal HMG-CoA reductase confirms that this C-terminal serine is the only major site phosphorylated in the intact enzyme by the AMP-activated protein kinase. The catalytic fragment of HMG-CoA reductase was also isolated from rat liver in the presence of protein phosphatase inhibitors under conditions where the enzyme is largely in the inactive form. HPLC, mass spectrometry and sequencing of the peptide containing Ser872 demonstrated that this site is high...
Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1988
The type-I protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase u and glycogen synthase ~betted in sites-3) as substrates) was S0-fold higher than that of the glycogen-bound enzyme. (2) The microson~ enzyme had a greater sensitivity to inhihitors-I and 2. (3) Release of the catalytic subun~t from the microsomal type-I phnsphatase by tLw~.¢ic digestion was accompanied by a ?.-fold increase in synthase phosphatase activity, whereas release ~! the catalytic subunit front the glycogen-bound enzyme decreased synthase phsophatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the miemsomes with 0~3 M NaCI, whereas little activity could be released from the glycogen fraction with salt. (5) The type-I phnsphatnse separated from glycogen by anion-exchange chromatowatm-~y could he rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCI) could not. These findings indicate that the synthnse igtOSlgtatase aetivity of the mierosomal enzyme is not explained by contamination with glycogen-lmead enzyme. The mlerosomal and gl~.Lmgen-associated ep_~y__mes__ ~_y eo_nt~in a common catalytic submit eomplexed to microsmual and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inln'bltor of the dephosighurylation of ribosomal protein 6,EIMG.CoAreductaseandglycogensyuthase,bythegly.˜−˜,en−asseciatedtype−Iwoteinls˜phatase.Bycontrast,thiophoslgtorylasea4idnotinin′bitthedqam˜′lgtorylatimtof6, EIMG.CoA reductase and glycogen syuthase, by the gly~.~-,en-asseciated type-I wotein l~sphatase. By contrast, thiophoslgtorylase a 4id not inin'bit the dqa~m'lgtorylatimt of 6,EIMG.CoAreductaseandglycogensyuthase,bythegly.˜−˜,en−asseciatedtype−Iwoteinls˜phatase.Bycontrast,thiophoslgtorylasea4idnotinin′bitthedqam˜′lgtorylatimtof6 or HMG,CoA redmtase by the mierosomal enzyme, although the delgtOS#~'ylation of glycogen synthase was inhibited. The/so for inhibition of synthase Igtosplm L~e activity by thio#mslgt~iase a catalysed by eilher the #yet~en.&~sociated or miemsmnal type-I phosphatases, or for inlgbition of 6phOslgmtaseactivityeatalysedbytheglycegen−associatedenzyme,wasdecreased20−foldto5−10nMinthewesenceofglycogen.Theresultssuggestthatthephysiologleailyrelevantinhibitoroftheglycogen−associatedtypedphosphataseisthephosphorylasea−glycogencomplex,andthatinhibitionofthemicrosemaltype−Iphosphatasebyphnsphurylaseaisunlikelytoplayaroleinthehormonalcontrolofcholesterolorproteinsynthesis.Proteinphosphatase−Iappearstobetheprincipal6 phOslgmtase activity eatalysed by the glycegen-associated enzyme, was decreased 20-fold to 5-10 nM in the wesence of glycogen. The results suggest that the physiologleaily relevant inhibitor of the glycogen-associated typed phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsemal type-I phosphatase by phnsphurylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-I appears to be the principal 6phOslgmtaseactivityeatalysedbytheglycegen−associatedenzyme,wasdecreased20−foldto5−10nMinthewesenceofglycogen.Theresultssuggestthatthephysiologleailyrelevantinhibitoroftheglycogen−associatedtypedphosphataseisthephosphorylasea−glycogencomplex,andthatinhibitionofthemicrosemaltype−Iphosphatasebyphnsphurylaseaisunlikelytoplayaroleinthehormonalcontrolofcholesterolorproteinsynthesis.Proteinphosphatase−Iappearstobetheprincipal6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.
Inactivation of rat liver HMG-CoA reductase phosphatases by polycarboxylic acids
Journal of Lipid Research, 1983
Incubation of the four purified HMG-CoA reductase phosphatases with the sodium salts of eleven polycarboxylic acids at concentrations of 40 mM, inactivated the enzymes to different degrees depending on the structure of the carboxylic acids. Maleate, malonate, oxalate, citrate, and hydroxymethylglutarate produced full inactivation at the concentration tested. When the four phosphatases were incubated with these acids, a concentrationdependent inactivation was observed. Fumarate, the trans isomer of maleate, produced little inactivation of the four phosphatases. Mevalonate did not inactivate at all. A relationship between those concentrations of acid that produced a 50% inactivation and the logarithm of the stability constant of Mg2+ or Mn2+ salts of polycarboxylic acids was observed. When reductase phosphatases were incubated with mixtures of polycarboxylic sodium salts and Mg2+ or Mn2+, an increase in the molar ratio divalent cation/carboxylic acid determined an increase in the four reductase phosphatase activities. The inactivating effect of citrate was on the phosphatases (high and low forms) and not on the substrates (MHG-CoA reductase, phosphorylase, and glycogen synthase). Reactivation of the citrate-inactivated phosphatases by Mn2+ and Mg2+ depended on the phosphorylated substrates, Mn2+ being the better activat0r.l It is concluded that HMECoA reductase phosphatases are metal1oenzymes.-Hegardt, F. G., G. Gil, and V. E. Calvet. Inactivation of rat liver HMG-CoA reductase phosphatases by polycarboxylic ac
Distinct type-2A protein phosphatases activate HMGCoA reductase and acetyl-CoA carboxylase in liver
FEBS Letters, 1997
Acetyl-CoA carboxylase and HMGCoA reductase are inactivated by the same AMP-activated protein kinase and are activated by type-2A protein phosphatase. To determine whether the same species of protein phosphatase-2A were involved, we studied the interconversion of acetyl-CoA carboxylase and HMGCoA reductase in isolated rat hepatocytes. We show that (i) these enzymes are differently regulated in hepatocytes and (ii) the species of type-2A protein phosphatase involved in their activation are different and can be separated by anion-exchange chromatography.
Molecular Biology of the Cell, 1996
In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmglp and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmglp were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmglp was localized in a pattern that resembled that of Hmglp when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmglp and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec6lp, was localized in the membranes induced by both Hmglp and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmglp:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmglp and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway. * Corresponding author. . Although cer-Abbreviations used: ER, endoplasmic reticulum; HMG, tamcett, includi the ER1988) Al BiP, and 3-hydroxy 3-methylglutaryl coenzyme A; HMGR, 3-hydroxy proteins including chaperone, 3-methylglutaryl coenzyme A reductase; DiOC6, 3,3'-dihexylox-protein disulfide isomerase, appear to be distributed acarbocyanineiodide; BiP, binding protein. throughout the entire ER (Sitia and Meldolesi, 1992), A.J. Koning et al. RWY410 MATa HMG1 HMG2 ura3-52 his3A200 ade2-101 lys2-801 met pAK266 (pGAL1-HMG1) This paper RWY446 MATa HMG1 HMG2 ura3-52 his3A200 ade2-101 lys2-801 met pRH134-2 (pGAL-HMG2) This paper RWY590 MATa HMG1 HMG2 leu2-3 leu2-112 prcl::LEU2 suc2A9 ura3-52 pMP375 (multicopy HMG2 Parrish et al., 1995 (same as with Loop G of HMG1) 591) RWY605 MATa HMG1 HMG2 leu2-3 leu2-112 prcl::LEU2 suc2A9 ura3-52 pJR360 (multicopy HMG2) Parrish et al., 1995 RWY621 MATa HMG1 HMG2 ura3-52 his3A200 ade2-101 lys2-801 met pCR425 (pGAL Hmgl:GFP) This paper RWY663 MATa HMG1 HMG2 ura3-52 his3A200 ade2-101 lys2-801 met pAK443 (pGAL Hmg2:GFP)