Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.27 (AMPK)
 6,299 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acetyl-CoA carboxylase has been purified from lactating rat mammary gland using a combination of ammonium sulphate and poly(ethyleneglycol) precipitations. The enzyme was purified from 35--70-fold with a yield of over 50%, the exact figures being difficult to estimate because of activation of the enzyme that occurs during the preparation. The preparation was homogeneous by the criterion of polyacrylamide gel electrophoresis in sodium dodecyl sulphate and had a single subunit of molecular weight 240,000, containing 1.02 +/- 0.04 molecules of biotin and 3.1 +/- 1.7 molecules of alkali-labile phosphate per subunit. The purified enzyme was phosphorylated and inactivated rapidly when incubated in the presence of [gamma 32P]ATP and magnesium ions with the purified catalytic subunit of cyclic-AMP-dependent protein kinase from rabbit skeletal muscle. Both phosphorylation and inactivation are blocked by the heat-stable protein inhibitor of cyclic-AMP-dependent protein kinase, and can be reversed by incubation with purified protein phosphatase-1 from rabbit skeletal muscle. The inactivation by the protein kinase and reactivation by the protein phosphatase correlate with the near-stoichiometric phosphorylation and dephosphorylation of site(s) located in a single tryptic peptide. Phosphorylation does not affect the Km for substrates, but brings about a twofold decrease in V and a twofold increase in the apparent dissociation constant for the allosteric activator, citrate. We also present evidence that the activation of rabbit mammary acetyl-CoA carboxylase by protein phosphatase-1 described previously [Hardie and Cohen (1979) FEBS Lett. 103, 333-338] is due to dephosphorylation at site(s) which are not phosphorylated by either cyclic-AMP-dependent protein kinase or acetyl-CoA carboxylase kinase-2. These results suggest that the rapid inactivation of acetyl-CoA carboxylase, and hence fatty acid synthesis, by adrenaline in adipose tissue, or glucagon in the liver, is due to phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase.
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PMID:Reversible phosphorylation and inactivation of acetyl-CoA carboxylase from lactating rat mammary gland by cyclic AMP-dependent protein kinase. 610 9

The catalytic subunit of cyclic AMP-dependent protein kinase stimulates the inactivation of acetyl-coenzyme A (CoA) carboxylase by acetyl-CoA carboxylase kinase. The stimulated inactivation of carboxylase is due to activation of carboxylase kinase by the catalytic subunit. Activation of carboxylase kinase activity is accompanied by the incorporation of 0.6 mol of phosphate per mole of carboxylase kinase. Addition of the regulatory subunit of cyclic AMP-dependent protein kinase prevents the activation of carboxylase kinase. Phosphorylation and activation of carboxylase kinase has no effect on the Km for ATP, but decreases the Km for acetyl-CoA carboxylase from 93 to 45 nM. Inactivation of carboxylase by the carboxylase kinase requires the presence of coenzyme A even when the activated carboxylase kinase is used. Acetyl-CoA carboxylase is not phosphorylated or inactivated by the catalytic subunit of cyclic AMP-dependent protein kinase.
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PMID:Phosphorylation and activation of acetyl-coenzyme A Carboxylase kinase by the catalytic subunit of cyclic AMP-dependent protein kinase. 631 99

Clostridium symbiosum pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP). The nucleotide binding site of this enzyme was labeled using the photoaffinity reagent [32P]-8-azidoadenosine 5'-triphosphate ([32P]-8-azidoATP). Subtilisin cleavage of the [alpha-32P]-8-azidoATP-photolabeled PPDK into domain-sized fragments, prior to SDS-PAGE analysis, allowed us to identify two sites of modification: one between residues 1 and 226 and the other between residues 227 and 334. Saturation of the ATP binding site with adenylyl imidodiphosphate afforded protection against photolabeling. Next, small peptide fragments of [gamma-32P]- 8-azidoATP-photolabeled PPDK were generated by treating the denatured protein with trypsin or alpha-chymotrypsin. A pair of overlapping radiolabeled peptide fragments were separated from the two digests, DMQDMEFTIEEGK (positions 318-330 in trypsin-treated PPDK) and RDMQDMEFTIEEGKL (positions 317-331 in alpha-chymotrypsin-treated PPDK), thus locating one of the positions of covalent modification. Next, catalysis by site-directed mutants generated by amino acid replacement of invariant residues of the PPDK N-terminal domain was tested. K163L, D168A, D170A, D175A, K177L, and G248I PPDK mutants retained substantial catalytic activity while G254I, R337L, and E323L PPDK mutants were inhibited. Comparison of the steady-state kinetic constants measured (at pH 6.8, 25 degrees C) for wild-type PPDK (kcat = 36 s-1, AMPK(m) = 7 microM, PP(i)K(m) = 70 microM, PEPK(m) = 27 microM) to those of R337L PPDK (kcat = 2 s-1, AMPK(m) = 85 microM, PP(i)K(m) = 3700 microM, PEPK(m) = 6 microM) and G254I PPDK (kcat = 0.1 s-1, AMPK(m) = 1300 microM, PP(i)K(m) = 1200 microM, PEPK(m) = 12 microM) indicated impaired catalysis of the nucleotide partial reaction (E.ATP.P(i) --> E-PP.AMP.P(i) --> E-P.AMP.PP(i) in these mutants. The single turnover reactions of [32P]PEP to [32P]E-P.pyruvate catalyzed by the PPDK mutants were shown to be comparable to those of wild-type PPDK. In contrast, the formation of [32P]E-PP/[32P]E-P in single turnover reactions of [beta-32P]ATP/P(i) was significantly inhibited. Finally, the location of the adenosine 5'-diphosphate binding site within the nucleotide binding domain of D-alanine-D-alanine ligase, a structural homologue of the PPDK N-terminal domain [Herzberg, O. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 2652-2657] indicates, by analogy, the location of the nucleotide binding site in PPDK. Residues G254, R337, and E323 as well as the site of photoaffinity labeling are located within this region.
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PMID:Determination of the nucleotide binding site within Clostridium symbiosum pyruvate phosphate dikinase by photoaffinity labeling, site-directed mutagenesis, and structural analysis. 867 15

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.
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PMID:Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. 1487 7

In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK). cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family. The identity of the enzyme was confirmed by immunoprecipitation of the active kinase from tobacco cells subjected to osmotic stress using antibodies raised against a peptide corresponding to the C-terminal sequence of the kinase predicted from the cloned cDNA. A detailed biochemical characterization of NtOSAK purified from stressed tobacco cells was performed. Our results show that NtOSAK is a calcium-independent Ser/Thr protein kinase. The sequence of putative phosphorylation sites recognized by NtOSAK, predicted by the computer program PREDIKIN, resembled the substrate consensus sequence defined for animal and yeast (Saccharomyces cerevisiae) AMPK/SNF1 kinases. Our experimental data confirmed these results, as various targets for AMPK/SNF1 kinases were also efficiently phosphorylated by NtOSAK. A range of protein kinase inhibitors was tested as potential modulators of NtOSAK, but only staurosporine, a rather nonspecific protein kinase inhibitor, was found to abolish the enzyme activity. In phosphorylation reactions, NtOSAK exhibited a preference for Mg(2+) over Mn(2+) ions and an inability to use GTP instead of ATP as a phosphate donor. The enzyme activity was not modulated by 5'-AMP. To our knowledge, these results represent the first detailed biochemical characterization of a kinase of the SnRK2 family.
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PMID:Biochemical characterization of the tobacco 42-kD protein kinase activated by osmotic stress. 1546 34

AMPK plays a central role in influencing fuel usage and selection. The aim of this study was to analyze the impact of low-dose AMP analog 5-aminoimidazole-4-carboxamide-1-beta-d-ribosyl monophosphate (ZMP) on whole body glucose turnover and skeletal muscle (SkM) glucose metabolism. Dogs were restudied after prior 48-h fatty acid oxidation (FA(OX)) blockade by methylpalmoxirate (MP; 5 x 12 hourly 10 mg/kg doses). During the basal equilibrium period (0-150 min), fasting dogs (n = 8) were infused with [3-(3)H]glucose followed by either 2-h saline or AICAR (1.5-2.0 mg x kg(-1) x min(-1)) infusions. SkM was biopsied at completion of each study. On a separate day, the same protocol was undertaken after 48-h in vivo FA(OX) blockade. The AICAR and AICAR + MP studies were repeated in three chronic alloxan-diabetic dogs. AICAR produced a transient fall in plasma glucose and increase in insulin and a small decline in free fatty acid (FFA). Parallel increases in hepatic glucose production (HGP), glucose disappearance (R(d tissue)), and glycolytic flux (GF) occurred, whereas metabolic clearance rate of glucose (MCR(g)) did not change significantly. Intracellular SkM glucose, glucose 6-phosphate, and glycogen were unchanged. Acetyl-CoA carboxylase (ACC approximately pSer(221)) increased by 50%. In the AICAR + MP studies, the metabolic responses were modified: the glucose was lower over 120 min, only minor changes occurred with insulin and FFA, and HGP and R(d tissue) responses were markedly attenuated, but MCR(g) and GF increased significantly. SkM substrates were unchanged, but ACC approximately pSer(221) rose by 80%. Thus low-dose AICAR leads to increases in HGP and SkM glucose uptake, which are modified by prior FA(ox) blockade.
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PMID:Impact of in vivo fatty acid oxidation blockade on glucose turnover and muscle glucose metabolism during low-dose AICAR infusion. 1677 28

A novel niacin-bound, chromium-based energy formula (EF; InterHealth Nutraceuticals, Benicia, CA) has been developed in conjunction with D-ribose, caffeine, ashwagandha extract (containing 5% withanolides), and selected amino acids. We have assessed the efficacy of oral administration of EF (40 mg x kg body wt(-1) x day(-1)) in male and female rats over a period of 90 consecutive days on the cardiovascular and pathophysiological functions in an isolated rat heart model. After 30, 60, and 90 days of treatment with EF, the hearts of male and female rats were subjected to 30 min of global ischemia followed by 2 h of reperfusion and were measured for myocardial ATP, creatine phosphate (CP), phosphorylated AMP kinase (p-AMPK), and heat shock proteins. Myocardial ATP and CP levels were increased in both male and female rats after EF treatment compared with the controls. Western blot analyses were performed to quantify the expression of stress-related proteins such as heat shock proteins (HSP-70, -32, and -25) and are found to be increased in both male and female rats after EF treatment. The p-AMPK level, which is a sensor for the energy state in various cell types, was also found to be increased after treatment with EF in both male and female rats. Aortic flow, maximum first derivative of developed pressure, left ventricular developed pressure, and infarct size were observed after ischemia-reperfusion and found to be significantly improved in EF-treated rats compared with control animals. Thus EF demonstrated long-term safety as well as exhibiting significant cardioprotective ability during ischemia and reperfusion injury by increased energy production, improved cardiac function, and reduced infarct size.
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PMID:Niacin-bound chromium enhances myocardial protection from ischemia-reperfusion injury. 1684 Jul 37

The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular ATP (adenosine triphosphate) and AMP concentrations. Here, we report crystal structures at 2.9 and 2.6 A resolution for ATP- and AMP-bound forms of a core alphabetagamma adenylate-binding domain from the fission yeast AMPK homolog. ATP and AMP bind competitively to a single site in the gamma subunit, with their respective phosphate groups positioned near function-impairing mutants. Unexpectedly, ATP binds without counterions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.
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PMID:Crystal structures of the adenylate sensor from fission yeast AMP-activated protein kinase. 1737 94

AICA riboside (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) has been extensively used in cells to activate the AMPK (AMP-activated protein kinase), a metabolic sensor involved in cell energy homoeostasis. In the present study, we investigated the effects of AICA riboside on mitochondrial oxidative; phosphorylation. AICA riboside was found to dose-dependently inhibit the oligomycin-sensitive JO2 (oxygen consumption rate) of isolated rat hepatocytes. A decrease in P(i) (inorganic phosphate), ATP, AMP and total adenine nucleotide contents was also observed with AICA riboside concentrations >0.1 mM. Interestingly, in hepatocytes from mice lacking both alpha1 and alpha2 AMPK catalytic subunits, basal JO2 and expression of several mitochondrial proteins were significantly reduced compared with wild-type mice, suggesting that mitochondrial biogenesis was perturbed. However, inhibition of JO2 by AICA riboside was still present in the mutant mice and thus was clearly not mediated by AMPK. In permeabilized hepatocytes, this inhibition was no longer evident, suggesting that it could be due to intracellular accumulation of Z nucleotides and/or loss of adenine nucleotides and P(i). ZMP did indeed inhibit respiration in isolated rat mitochondria through a direct effect on the respiratory-chain complex I. In addition, inhibition of JO2 by AICA riboside was also potentiated in cells incubated with fructose to deplete adenine nucleotides and P(i). We conclude that AICA riboside inhibits cellular respiration by an AMPK-independent mechanism that likely results from the combined intracellular P(i) depletion and ZMP accumulation. Our data also demonstrate that the cellular effects of AICA riboside are not necessarily caused by AMPK activation and that their interpretation should be taken with caution.
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PMID:AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside. 1732 22

The tumour suppressor LKB1 plays a critical role in cell proliferation, polarity and energy metabolism. LKB1 is a Ser/Thr protein kinase that is associated with STRAD and MO25 in vivo. Here, we describe the individual expression of the three components of the LKB1 complex using monocistronic vectors and their co-expression using tricistronic vectors that were constructed from monocistronic vectors using a fully modular cloning approach. The data show that among the three individually expressed components of the LKB1 complex, only MO25alpha can be expressed in soluble form, whereas the other two, LKB1 and STRADalpha are found almost exclusively in inclusion bodies. However, using the tricistronic vector system, functional LKB1-MO25alpha-STRADalpha complex was expressed and purified from soluble extracts by sequential immobilized-metal affinity and heparin chromatography, as shown by Western blotting using specific antibodies. In size exclusion chromatography, MO25alpha and STRADalpha exactly co-elute with LKB1 with an apparent molecular weight of the heterotrimeric complex of 160 kDa. The specific activity in the peak fraction of the size exclusion chromatography was 250 U/mg at approximately 25% purity. As shown by autoradiography, LKB1 and STRADalpha, both strongly autophosphorylate in vitro. Moreover, recombinant LKB1 complex activates AMPK by phosphorylation of the alpha-subunit at the Thr-172 site as shown (i) by Western blotting using phospho-specific antibodies after LKB1-dependent phosphorylation, (ii) by LKB1-dependent incorporation of radioactive phosphate into the alpha-subunit of kinase dead AMPK heterotrimer, and (iii) by activity determination of AMPK. Functional mammalian LKB1 complex is constitutively active, and when enriched from bacteria should prove to be a valuable tool for studying its molecular function and regulation.
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PMID:Co-expression of LKB1, MO25alpha and STRADalpha in bacteria yield the functional and active heterotrimeric complex. 1787 8


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