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The Journal of Biological Chemistry Feb 2011The anaerobic acetogenic bacterium Acetobacterium woodii employs a novel type of Na(+)-motive anaerobic respiration, caffeate respiration. However, this respiration is...
The anaerobic acetogenic bacterium Acetobacterium woodii employs a novel type of Na(+)-motive anaerobic respiration, caffeate respiration. However, this respiration is at the thermodynamic limit of energy conservation, and even worse, in the first step, caffeate is activated by caffeyl-CoA synthetase, which hydrolyzes ATP to AMP and pyrophosphate. Here, we have addressed whether or not the energy stored in the anhydride bond of pyrophosphate is conserved by A. woodii. Inverted membrane vesicles of A. woodii have a membrane-bound pyrophosphatase that catalyzes pyrophosphate hydrolysis at a rate of 70-120 milliunits/mg of protein. Pyrophosphatase activity was dependent on the divalent cation Mg(2+). In addition, activity was strictly dependent on Na(+) with a K(m) of 1.1 mM. Hydrolysis of pyrophosphate was accompanied by (22)Na(+) transport into the lumen of the inverted membrane vesicles. Inhibitor studies revealed that (22)Na(+) transport was primary and electrogenic. Next to the Na(+)-motive ferredoxin:NAD(+) oxidoreductase (Fno or Rnf), the Na(+)-pyrophosphatase is the second primary Na(+)-translocating enzyme in A. woodii.
Topics: Acetobacterium; Anaerobiosis; Bacterial Proteins; Catalysis; Cation Transport Proteins; Pyrophosphatases; Sodium
PubMed: 21173152
DOI: 10.1074/jbc.M110.192823 -
The Journal of Biological Chemistry Dec 1987The bisphosphatase domain of the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to exhibit a structural similarity to yeast...
The bisphosphatase domain of the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to exhibit a structural similarity to yeast phosphoglycerate mutase and human red blood cell 2,3-bisphosphoglycerate mutase including very similar active site sequences with a histidyl residue being involved in phospho group transfer. The liver bifunctional enzyme was found to catalyze the hydrolysis of glycerate 1,3-bisphosphate to glycerate 3-phosphate and inorganic phosphate. The Km for glycerate 1,3-bisphosphate was 320 microM and the Vmax was 11.5 milliunits/mg. Incubation of the rat liver enzyme with [1-32P]glycerate 1,3-bisphosphate resulted in the formation of a phosphoenzyme intermediate, and the labeled amino acid was identified as 3-phosphohistidine. Tryptic and endoproteinase Lys-C peptide maps of the 32P-phosphoenzyme labeled either with [2-32P]fructose 2,6-bisphosphate or [1-32P]glycerate 1,3-bisphosphate revealed that 32P-radioactivity was found in the same peptide, proving that the same histidyl group accepts phosphate from both substrates. Fructose 2,6-bisphosphate inhibited competitively the formation of phosphoenzyme from [1-32P]glycerate 1,3-bisphosphate. Effectors of fructose-2,6-bisphosphatase also inhibited phosphoenzyme formation. Substrates and products of phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase also modulated the activities of the bifunctional enzyme. These results demonstrate that, in addition to a structural homology, the bisphosphatase domain of the bifunctional enzyme has a functional similarity to phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase and support the concept of an evolutionary relationship between the three enzyme activities.
Topics: Adenosine Triphosphate; Bisphosphoglycerate Mutase; Diphosphoglyceric Acids; Erythrocytes; Hot Temperature; Humans; Isomerases; Kinetics; Liver; Peptide Mapping; Phosphofructokinase-2; Phosphoglycerate Mutase; Phosphoric Monoester Hydrolases; Phosphotransferases; Structure-Activity Relationship
PubMed: 2824507
DOI: No ID Found -
The Journal of Clinical Endocrinology... Nov 2012Data on thyroid function in the oldest old are sparse, and existing studies show conflicting evidence on the relationship between thyroid function and mortality in this...
CONTEXT
Data on thyroid function in the oldest old are sparse, and existing studies show conflicting evidence on the relationship between thyroid function and mortality in this age group.
OBJECTIVE
We describe longitudinal changes in thyroid function in a cohort of elderly individuals and determine the relationship between thyroid function and mortality.
DESIGN, SETTING, AND PARTICIPANTS
Eight hundred forty-three participants in the Cardiovascular Health Study All Stars Study who were not taking thyroid medications and had thyroid function testing in 2005-2006 (mean age 85 yr).
MAIN OUTCOME MEASURE
Thyroid-stimulating hormone (TSH), free T(4) (FT4), total T(3), and thyroid peroxidase antibody status were measured in 1992-1993 and 2005-2006. Deaths were ascertained through February 2011.
RESULTS
There was a statistically significant 13% increase in TSH, 1.7% increase in FT4, and 13% decrease in total T(3) over the 13-yr period. Two hundred eighty-seven deaths occurred over a median follow-up of 5.1 yr. There was no association between subclinical hypothyroidism[hazard ratio (HR) 0.97, 95% confidence interval (CI) 0.66-1.43], TSH level (HR per milliunits per liter 0.94, 95% CI 0.88-1.01), or persistent thyroid peroxidase antibody positivity (HR 1.09, 95% CI 0.62-1.92), and death. However, FT4 was positively associated with death (HR per nanograms per deciliter 2.57, 95% CI 1.32-5.02).
CONCLUSIONS
TSH increased over time in these older individuals. This elevation was not associated with increased or decreased mortality, although higher FT4 levels were associated with death. These findings raise concern for treatment of mild elevations of TSH in advanced age. Further studies are needed to determine the potential benefit of treating age-related changes in thyroid function.
Topics: Aged, 80 and over; Female; Humans; Longitudinal Studies; Male; Thyroid Diseases; Thyroid Function Tests; Thyroid Gland; Thyrotropin; Thyroxine; Triiodothyronine
PubMed: 22879629
DOI: 10.1210/jc.2012-2481 -
The Journal of Biological Chemistry Feb 1982Phenylephrine (1.5 x 10(-6) M) administered to perfused livers from fed rats gave rise to a rapid, parallel increase in oxygen uptake and glucose output. The time of...
Phenylephrine (1.5 x 10(-6) M) administered to perfused livers from fed rats gave rise to a rapid, parallel increase in oxygen uptake and glucose output. The time of onset for oxygen uptake was 9.9 +/- 0.4 s following phenylephrine administration, and immediately preceded glucose output which occurred at 11.6 +/- 0.5 s. Near-maximal effects were observed 50 s following alpha-agonist treatment. Both responses appear to be mediated by alpha- 1-adrenergic receptors. The mitochondrial respiratory chain blockers antimycin A and rotenone, inhibited the alpha-agonist-induced oxygen uptake and glycogenolytic responses at inhibitor concentrations similar to those required to block uncoupler-stimulated respiration in the intact perfused liver. Oligomycin and carboxyatractyloside also inhibited the phenylephrine-induced respiratory response. Vasopressin (1 milliunit/ml), and angiotensin II (6 x 10(-9) M) had effects similar to phenylephrine in the perfused liver which also were prevented by the prior administration of antimycin A and rotenone. In contrast, glucagon-induced (10(-8) M) glycogenolysis proceeded in the absence of large changes in respiration, was slower in onset (26.1 +/- 4.2 s following hormone administration), and was not inhibited by mitochondrial respiratory blockers. These data indicate that glycogenolysis induced by alpha-adrenergic agonists, vasopressin, and angiotensin II is associated with a large increase in mitochondrial respiration, that may play a role in a general, as yet undefined mechanism whereby these agents stimulate glycogenolysis in rat liver.
Topics: Animals; Antimycin A; Dinitrophenols; Female; Glucagon; Kinetics; Liver; Liver Glycogen; Oxygen Consumption; Phenylephrine; Prazosin; Rats; Rats, Inbred Strains; Rotenone; Yohimbine
PubMed: 7056751
DOI: No ID Found -
The Journal of Biological Chemistry Apr 2002Based on the previous report of McCord and co-workers (Crow, J. P., Beckman, J. S., and McCord, J. M. (1995) Biochemistry 34, 3544-3552), the zinc dithiolate active site...
Based on the previous report of McCord and co-workers (Crow, J. P., Beckman, J. S., and McCord, J. M. (1995) Biochemistry 34, 3544-3552), the zinc dithiolate active site of alcohol dehydrogenase (ADH) has been studied as a target for cellular oxidants. In the nitrogen monoxide ((*NO)/superoxide (O(2)) system, an equimolar generation of both radicals under peroxynitrite (PN) formation led to rapid inactivation of ADH activity, whereas hydrogen peroxide and ( small middle dot)NO alone reacted too slowly to be of physiological significance. 3-Morpholino sydnonimine inactivated the enzyme with an IC(50) value of 250 nm; the corresponding values for PN, hydrogen peroxide, and (*NO) were 500 nm, 50 microm, and 200 microm. When superoxide was generated at low fluxes by xanthine oxidase, it was quite effective in ADH inactivation (IC(50) (XO) approximately 1 milliunit/ml). All inactivations were accompanied by zinc release and disulfide formation, although no strict correlation was observed. From the two zinc thiolate centers, only the zinc Cys(2)His center released the metal by oxidants. The zinc Cys(4) center was also oxidized, but no second zinc atom could be found with 4-(2-pyridylazo)resorcinol (PAR) as a chelating agent except under denaturing conditions. Surprisingly, the oxidative actions of PN were abolished by a 2-3-fold excess of (*)NO under generation of a nitrosating species, probably dinitrogen trioxide. We conclude that in cellular systems, low fluxes of (*)NO and O(2) generate peroxynitrite at levels effective for zinc thiolate oxidations, facilitated by the nucleophilic nature of the complexed thiolate group. With an excess of (*)NO, the PN actions are blocked, which may explain the antioxidant properties of (*)NO and the mechanism of cellular S-nitrosations.
Topics: Animals; Biochemical Phenomena; Biochemistry; Cattle; Chelating Agents; Chromatography, High Pressure Liquid; Chromogenic Compounds; Disulfides; Dose-Response Relationship, Drug; Free Radicals; Hydrazines; Hydrogen Peroxide; Models, Chemical; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitrogen; Nitrogen Oxides; Oxygen; Peroxynitrous Acid; Protein Binding; Resorcinols; Spectrophotometry; Spectrum Analysis, Raman; Spermine; Sulfhydryl Compounds; Superoxides; Time Factors; Ultraviolet Rays; Xanthine Oxidase; Zinc
PubMed: 11805115
DOI: 10.1074/jbc.M111988200 -
The Journal of Biological Chemistry Apr 1996The sphingomyelin pathway is a newly described signal transduction pathway mediating the action of several cytokines including tumor necrosis factor-alpha (TNF). TNF was...
The sphingomyelin pathway is a newly described signal transduction pathway mediating the action of several cytokines including tumor necrosis factor-alpha (TNF). TNF was recently shown to interfere with insulin-induced tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1). In this work we examined the possible effect of direct activation of the sphingomyelin pathway on insulin-induced tyrosine phosphorylation of IRS-1. Incubation of the insulin-sensitive rat hepatoma Fao cells with bacterial sphingomyelinase (SMase) that causes membrane hydrolysis of sphingomyelin led to a time- and dose-dependent decrease in insulin-induced tyrosine phosphorylation of IRS-1. The effect was apparent after 10 min of incubation and with a dose of 10 milliunits/ml SMase. It was not associated with a decrease in insulin receptor autophosphorylation. In addition, SMase treatment interrupted the association of the 85-kDa catalytic subunit of phosphatidylinositol 3-kinase with IRS-1. A similar impact on IRS-1 tyrosine phosphorylation was observed after addition of cell-permeable ceramide analogs (C2 and C6). Comparable changes in IRS-1 tyrosine phosphorylation and electrophoretic mobility were found after exposure of cells to either TNF, SMase, or ceramide. Our findings suggest that TNF may utilize the sphingomyelin pathway in its effect on the insulin-stimulated tyrosine phosphorylation of IRS-1.
Topics: Animals; Ceramides; Enzyme Inhibitors; Insulin; Insulin Receptor Substrate Proteins; Liver Neoplasms, Experimental; Phosphoproteins; Phosphotyrosine; Rats; Receptor, Insulin; Signal Transduction; Sphingomyelin Phosphodiesterase; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha
PubMed: 8626623
DOI: 10.1074/jbc.271.17.9895 -
The Journal of Clinical Investigation Jun 1993Absence of dietary carbohydrate decreases both activities of intestinal brush border sucrase-alpha-dextrinase. We examined the molecular mechanism causing this decrease.... (Comparative Study)
Comparative Study
Absence of dietary carbohydrate decreases both activities of intestinal brush border sucrase-alpha-dextrinase. We examined the molecular mechanism causing this decrease. Adult rats were fed chow (70% CHO) or matched carbohydrate-free (CHO-free) diet for 7 d. Sucrase activity decreased by 50% in whole homogenates and brush borders. Enzyme kinetics revealed no change in sucrose affinity (CHO-free Km = 18 mM, chow Km = 21 mM), but fewer active sites (CHO-free Vmax = 2,720, chow Vmax = 5,000 mumol/min per g protein). Intraintestinal pulse-labeling of [35S]methionine in vivo revealed no differences in incorporation into sucrase. Immunoreactive sucrase protein, assayed by ELISA and rocket immunoelectrophoresis, increased twofold per milliunit of sucrase enzymatic activity in CHO-free jejunum. Total immunosucrase (St), the sum of active and inactive enzyme (St = Sa+Si), was unchanged with carbohydrate withdrawal, but > 50% of the sucrase protein became inactive. SDS-PAGE of sucrase immunoprecipitates revealed alteration of alpha, beta, and gamma subunits in CHO-free animals: (a) alpha and beta subunits migrated farther (mass change--2 kD); and (b) the alpha subunit became diffuse or was a doublet and was less abundant than the beta subunit. Rather than representing loss of sucrase protein, the decline in sucrase activity is achieved with structural subunit changes, probably involving postinsertional processing.
Topics: Animals; Carbohydrates; Dietary Carbohydrates; Immunoassay; Kinetics; Male; Microvilli; Protein Precursors; Protein Processing, Post-Translational; Rats; Rats, Wistar; Sucrase; Sucrase-Isomaltase Complex
PubMed: 8514885
DOI: 10.1172/JCI116520 -
The Journal of Biological Chemistry Oct 1999The impact of increased GlcN availability on insulin-stimulated p85/p110 phosphatidylinositol 3-kinase (PI3K) activity in skeletal muscle was examined in relation to...
The impact of increased GlcN availability on insulin-stimulated p85/p110 phosphatidylinositol 3-kinase (PI3K) activity in skeletal muscle was examined in relation to GlcN-induced defects in peripheral insulin action. Primed continuous GlcN infusion (750 micromol/kg bolus; 30 micromol/kg.min) in conscious rats limited both maximal stimulation of muscle PI3K by acute insulin (I) (1 unit/kg) bolus (I + GlcN = 1.9-fold versus saline = 3.3-fold above fasting levels; p < 0.01) and chronic activation of PI3K following 3-h euglycemic, hyperinsulinemic (18 milliunits/kg.min) clamp studies (I + GlcN = 1.2-fold versus saline = 2.6-fold stimulation; p < 0.01). To determine the time course of GlcN-induced defects in insulin-stimulated PI3K activity and peripheral insulin action, GlcN was administered for 30, 60, 90, or 120 min during 2-h euglycemic, hyperinsulinemic clamp studies. Activation of muscle PI3K by insulin was attenuated following only 30 min of GlcN infusion (GlcN 30 min = 1.5-fold versus saline = 2.5-fold stimulation; p < 0.05). In contrast, the first impairment in insulin-mediated glucose uptake (Rd) developed following 110 min of GlcN infusion (110 min = 39.9 +/- 1.8 versus 30 min = 42.8 +/- 1.4 mg/kg.min, p < 0.05). However, the ability of insulin to stimulate phosphatidylinositol 3,4, 5-trisphosphate production and to activate glycogen synthase in skeletal muscle was preserved following up to 180 min of GlcN infusion. Thus, increased GlcN availability induced (a) profound and early inhibition of proximal insulin signaling at the level of PI3K and (b) delayed effects on insulin-mediated glucose uptake, yet (c) complete sparing of insulin-mediated glycogen synthase activation. The pattern and time sequence of GlcN-induced defects suggest that the etiology of peripheral insulin resistance may be distinct from the rapid and marked impairment in insulin signaling.
Topics: Animals; Blood Glucose; Drug Interactions; Enzyme Activation; Glucosamine; Glucose Clamp Technique; Glycolysis; Insulin; Male; Muscle, Skeletal; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Rats; Rats, Sprague-Dawley; Uridine Diphosphate N-Acetylglucosamine
PubMed: 10531330
DOI: 10.1074/jbc.274.44.31312 -
The Journal of Biological Chemistry May 2000CCAAT/enhancer-binding protein beta (C/EBPbeta) controls gene transcription and metabolic processes in a variety of insulin-sensitive tissues; however, its role in...
CCAAT/enhancer-binding protein beta (C/EBPbeta) controls gene transcription and metabolic processes in a variety of insulin-sensitive tissues; however, its role in regulating insulin responsiveness in vivo has not been investigated. We performed hyperinsulinemic-euglycemic clamps in awake, non-stressed, chronically catheterized adult mice homozygous for a deletion in the gene for C/EBPbeta (C/EBPbeta(-/-)). Fasting plasma insulin, glucose, and free fatty acid (FFA) levels were significantly lower in C/EBPbeta(-/-) mice compared with wild-type (WT) controls. Acute hyperinsulinemia (4 h) suppressed hepatic glucose production, phosphoenolpyruvate carboxykinase mRNA, and plasma FFA to a similar extent in WT and C/EBPbeta(-/-) mice, suggesting that C/EBPbeta deletion does not alter the metabolic and gene regulatory response to insulin in liver and adipose tissue. In contrast, using submaximal (5 milliunits/kg/min) and maximal (20 milliunits/kg/min) insulin infusions, whole-body glucose disposal was 77% (p < 0.01) and 33% (p < 0.05) higher in C/EBPbeta(-/-) mice, respectively, compared with WT mice. Maximal insulin-stimulated 3-O-methylglucose uptake in isolated soleus muscle was 54% greater in C/EBPbeta(-/-) mice (p < 0.05). Furthermore, insulin-stimulated insulin receptor and Akt Ser(473) phosphorylation and phosphatidylinositol 3-kinase activity were 1.6-2.5-fold greater in skeletal muscle from C/EBPbeta(-/-) mice compared with WT mice. The level of insulin receptor substrate-1 protein was increased 2-fold in skeletal muscle from C/EBPbeta(-/-) mice. These results demonstrate that C/EBPbeta deletion decreases plasma FFA levels and increases insulin signal transduction specifically in skeletal muscle, and both contribute to increased whole-body insulin sensitivity.
Topics: Adipose Tissue; Animals; CCAAT-Enhancer-Binding Proteins; DNA-Binding Proteins; Female; Gene Expression Regulation; Glucose; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Liver; Mice; Mice, Knockout; Muscle, Skeletal; Nuclear Proteins; Phosphoproteins; Signal Transduction
PubMed: 10747954
DOI: 10.1074/jbc.m000764200 -
The Journal of Biological Chemistry Aug 1981The effect of natural "activation factor" and synthetic fructose-2,6-P2 on the allosteric kinetic properties of liver and muscle phosphofructokinases was investigated....
The effect of natural "activation factor" and synthetic fructose-2,6-P2 on the allosteric kinetic properties of liver and muscle phosphofructokinases was investigated. Both synthetic and natural fructose-2,6-P2 show identical effects on the allosteric kinetic properties of both enzymes. Fructose-2,6-P2 counteracts inhibition by ATP and citrate and decreases the Km for fructose-6-P. This fructose ester also acts synergistically with AMP in releasing ATP inhibition. The Km values of liver and muscle phosphofructokinase for fructose-2,6-P2 in the presence of 1.25 mM ATP are 12 milliunits/ml (or 24 nM) and 5 milliunits/ml (or 10 nM), respectively. At near physiological concentrations of ATP (3 mM) and fructose-6-P (0.2 mM), however, the Km values for fructose-2,6-P2 are increased to 12 microM and 0.8 microM for liver and muscle enzymes, respectively. Thus, fructose-2,6-P2 is the most potent activator of the enzyme compared to other known activators such as fructose-1,6-P2. The rates of the reaction catalyzed by the enzymes under the above conditions are nonlinear: the rates decelerate in the absence or in the presence of lower concentrations of fructose-2,6-P2, but the rates become linear in the presence of higher concentrations of fructose-2,6-P2. Fructose-2,6-P2 also protects phosphofructokinase against inactivation by heat. Fructose-2,6-P2, therefore, may be the most important allosteric effector in regulation of phosphofructokinase in liver as well as in other tissues.
Topics: Adenosine Monophosphate; Adenosine Triphosphate; Allosteric Regulation; Animals; Citrates; Enzyme Activation; Fructosediphosphates; Hexosediphosphates; Kinetics; Liver; Muscles; Organ Specificity; Phosphofructokinase-1; Rats
PubMed: 6455423
DOI: No ID Found