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PubMed1976

Enzymes of carbohydrate metabolism in four human species of Leishmania: a comparative survey.

The occurrence and levels of activity of various enzymes of carbohydrate catabolism in culture forms (promastigotes) of 4 human species of Leishmania (L. brasiliensis, L. donovani, L. mexicana, and L. tropica) were compared. These organisms possess enzymes of the Embden-Meyerhof pathway but lack lactate dehydrogenase. No evidence could be found for the production of lactic acid by growing cultures and lactic acid could not be detected either in cell-free preparations or after incubation of cell-free extracts with pyruvate and NADH under appropriate conditions. All 4 species possess alpha-glycerophosphate dehydrogenase and alpha-glycerophosphate phosphatase which together could regenerate NAD, thus compensating for the absence of lactate dehydrogenase. The oxidative and nonoxidative reactions of the hexose monophosphate pathway are present in all 4 species. Cell-free extracts have pyruvate dehydrogenase activity which allows the entry of pyruvate into and its subsequent oxidation through the tricarboxylic acid cycle. All enzymes of this cycle, including a thiamine pyrophosphate dependent alpha-ketoglutarate dehydrogenase, are present. Both NAD and NADP-linked malate dehydrogenase activities are present. The isocitrate dehydrogenase is NADP specific. There is an active glutamate dehydrogenase which could compete with alpha-ketoglutarate dehydrogenase for the common substrate (alpha-ketoglutarate). Replenishment of C4 acids is accomplished by heterotrophic CO2 fixation catalyzed by pyruvate carboxylase. All 4 species have high levels of NADH oxidase activity. Several enzymes thus far not found in any species of Leishmania have been demonstrated. These are: phosphoglucose isomerase, triose phosphate isomerase, fructose-1, 6-diphosphatase, 3-phosphoglycerate kinase, enolase, alpha-glycerophosphate dehydrogenase, alpha-glycerophosphate phosphatase, pyruvate dehydrogenase complex, citrate synthase, aconitase, alpha-ketoglutarate dehydrogenase, glutamate dehydrogenase, and NADH oxidase.

Animals↗
PubMed1985

Unidirectional actions of insulin and Ca2+-dependent hormones on adipocyte pyruvate dehydrogenase.

Norepinephrine and epinephrine, in the presence of the beta-adrenergic antagonist propranolol (10(-5) M), stimulated adipocyte pyruvate dehydrogenase at low concentrations but inhibited the enzyme at higher concentrations. The alpha-adrenergic agonist, phenylephrine, rapidly stimulated pyruvate dehydrogenase activity in a dose-dependent manner with maximal stimulation observed at 10(-6) M. The stimulation of pyruvate dehydrogenase by phenylephrine was mediated via alpha 1-receptors. Inhibition of pyruvate dehydrogenase by catecholamines was mediated via beta-adrenergic receptors, since the beta-agonist, isoproterenol, and dibutyryl cAMP produced similar effects. Like insulin, alpha-adrenergic agonists increased the active form of pyruvate dehydrogenase without changing the total enzyme activity and cellular ATP concentration. The effects induced by maximally effective concentrations of insulin and alpha-adrenergic agonists were nonadditive. The ability of phenylephrine and methoxamine to stimulate pyruvate dehydrogenase and phosphorylase and to inhibit glycogen synthase was not affected by the removal of extracellular Ca2+. Similarly, the stimulation of pyruvate dehydrogenase and glycogen synthase by insulin was also observed under the same conditions. However, when intracellular adipocyte Ca2+ was depleted by incubating cells in a Ca2+-free buffer containing 1 mM ethylene glycol bis(beta-amino-ethyl ether)-N,N,N' -tetraacetic acid, the actions of alpha-adrenergic agonists, but not insulin, on pyruvate dehydrogenase were completely abolished. Vasopressin and angiotensin II also stimulated pyruvate dehydrogenase in a dose-dependent manner with enhancement of glucose oxidation and lipogenesis. Our results demonstrate that the Ca2+ -dependent hormones stimulate pyruvate dehydrogenase and lipogenesis in isolated rat adipocytes, and the action is dependent upon intracellular, but not extracellular, Ca2+.

Adipose Tissue↗
PubMed1982

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.

Acetates↗
PubMedDate not supplied

Age-related alterations of skeletal muscle metabolism by intermittent hypoxia and TRH-analogue treatment.

The characteristics of the energy metabolism were evaluated in the gastrocnemius muscle from 3- and 24-month-old rats in normoxia or subjected to either mild or severe chronic (4 weeks) intermittent normobaric hypoxia. Furthermore, 4-week treatment with saline or the TRH-analogue posatireline was performed. The muscular concentration of the following metabolites related to the energy metabolism was evaluated: glycogen, glucose, glucose 6-phosphate, pyruvate, lactate, lactate-to-pyruvate ratio; citrate, alpha-ketoglutarate, succinate, malate; aspartate, glutamate, alanine; ammonia; ATP, ADP, AMP, creatine phosphate; energy charge potential. Furthermore the maximum rate of the following muscular enzymes was evaluated: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase; citrate synthase, malate dehydrogenase; total NADH cytochrome c reductase; cytochrome oxidase. The age-related decrease in muscular glucose 6-phosphate, pyruvate and alanine concentrations and increase in citrate concentration were consistent with the age-related decreased hexokinase and increased citrate synthase activities. Ageing was characterized by a decrease in muscular creatine phosphate concentration, while the energy mediators and the energy charge potential were unchanged. The chronic (4 weeks) intermittent normobaric mild and severe hypoxia-induced alterations of the components in the anaerobic glycolytic pathway, tricarboxylic acid cycle and energy storage, that were magnified in the skeletal muscle from the oldest animals. The effect of the chronic treatment with the TRH-analogue posatireline suggests that the action of central nervous system-acting drugs could also be related to their direct influence on the muscular biochemical mechanisms related to the energy transduction.

Aging↗
PubMed1997

The Role of Sugars, Hexokinase, and Sucrose Synthase in the Determination of Hypoxically Induced Tolerance to Anoxia in Tomato Roots.

Hypoxic pretreatment of tomato (Lycopersicon esculentum M.) roots induced an acclimation to anoxia. Survival in the absence of oxygen was improved from 10 h to more than 36 h if external sucrose was present. The energy charge value of anoxic tissues increased during the course of hypoxic acclimation, indicating an improvement of energy metabolism. In acclimated roots ethanol was produced immediately after transfer to anoxia and little lactic acid accumulated in the tissues. In nonacclimated roots significant ethanol synthesis occurred after a 1-h lag period, during which time large amounts of lactic acid accumulated in the tissues. Several enzyme activities, including that of alcohol dehydrogenase, lactate dehydrogenase, pyruvate decarboxylase, and sucrose synthase, increased during the hypoxic pretreatment. In contrast to maize, hexokinase activities did not increase and phosphorylation of hexoses was strongly inhibited during anoxia in both kinds of tomato roots. Sucrose, but not glucose or fructose, was able to sustain glycolytic flux via the sucrose synthase pathway and allowed anoxic tolerance of acclimated roots. These results are discussed in relation to cytosolic acidosis and the ability of tomato roots to survive anoxia.

Journal Article↗
PubMed2004

Metabolic characteristics of experimental free vascularized canine gracilis muscle transfers.

A canine gracilis model was used to study muscle energy metabolism and enzyme activities after free vascularized muscle transfer. Fifteen male mongrel dogs underwent orthotopic, free transfer of the left gracilis with microneurovascular anastomosis. After a minimum of 10 months' recovery, muscle biopsy specimens were obtained from the transfers and the contralateral controls and analyzed for relative fiber type areas and maximum activities of phosphorylase, hexokinase, phosphofructokinase, glycerol-3-phosphate dehydrogenase (GPDH), pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, 3-hydroxyacyl coenzyme A dehydrogenase (HAD), and creatine phosphokinase. Biopsy specimens obtained before and after a 10 minute, 20-Hz contraction were analyzed for glucose, glycogen, glycolytic intermediates, phosphocreatine, total creatine, and adenine nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, and inosine). There was no significant transfer versus control difference in type I relative fiber area (45 +/- 4 percent versus 44 +/- 3 percent). Total creatine was significantly reduced in the transferred muscles relative to control (83.1 +/- 3.0 mmol/kg versus 100.6 +/- 5.1 mmol/kg dry weight). Maximal activities of phosphorylase, pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, HAD, and creatine phosphokinase were diminished in transfers relative to controls, although hexokinase activity was significantly higher in the freely transferred gracilis muscles. During the 20-Hz contraction, muscle transfers produced less force initially, although the force/time integral over the 10-minute stimulation was similar in transfers (277 +/- 25 N/g/second) and controls (272 +/- 24 N/g/second). The contraction was associated with significant glvcogen use and lactate accumulation in both transfers and controls, although this was less pronounced for the transfers. Glycolytic flux appeared muted in the transfers relative to controls. Significant, similar high-energy phosphagen reductions and inosine monophosphate accumulation were noted during the contraction in both groups. Contractile activity is associated with the expected pattern of muscle metabolite changes following free vascularized transfer, indicating the components of cellular energy metabolism are not qualitatively altered after microneurovascular muscle transfer. In contrast, quantitative differences suggest that free vascularized muscle transfer can be associated with a muscle enzyme profile consistent with deconditioning and the presence of denervated muscles fibers in the absence of fiber type profile changes.

Animals↗
PubMed1979

The role of calcium ion as a mediator of the effects of angiotensin II, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes.

Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis and gluconeogenesis via a cyclic AMP-independent mechanism that requires calcium ion. The present study explores the possibility that angiotensin II and vasopressin control the activity of regulatory enzymes in carbohydrate metabolism through Ca2+-dependent changes in their state of phosphorylation. Intact hepatocytes labeled with [32P]PO43- were stimulated with angiotensin II, glucagon, or vasopressin and 30 to 33 phosphorylated proteins resolved from the cytoplasmic fraction of the cell by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. Treatment of the cells with angiotensin II or vasopressin increased the phosphorylation of 10 to 12 of these cytosolic proteins without causing measurable changes in cyclic AMP-dependent protein kinase activity. Glucagon stimulated the phosphorylation of the same set of 11 to 12 proteins through a marked increase in cyclic AMP-dependent protein kinase activity. The molecular weights of three of the protein bands whose phosphorylation was increased by these hormones correspond to the subunit molecular weights of phosphorylase (Mr = 93,000), glycogen synthase (Mr = 85,000), and pyruvate kinase (Mr = 61,000). Two of these phosphoprotein bands were positively identified as phosphorylase and pyruvate kinase by affinity chromatography and immunoprecipitation, respectively. Incubation of hepatocytes in a Ca2+-free medium completely abolished the effects of angiotensin II and vasopressin on protein phosphorylation but did not alter those of glucagon. Treatment of hepatocytes with angiotensin II, glucagon, or vasopressin stimulated phosphorylase activity by 250 to 260%, inhibited glycogen synthase activity by 50%, and inhibited pyruvate kinase activity by 30 to 35% (peptides) to 70% (glucagon). The effects of angiotensin II and vasopressin on the activity of all three enzymes were completely abolished if the cells were incubated in a Ca2+-free medium while those of glucagon were not altered. The results imply that angiotensin II, catecholamines, and vasopressin control hepatic carbohydrate metabolism through a Ca2+-requiring, cyclic AMP-independent pathway that leads to the phosphorylation of important regulatory enzymes.

Angiotensin II↗
PubMed1993

Expression and lipoylation in Escherichia coli of the inner lipoyl domain of the E2 component of the human pyruvate dehydrogenase complex.

The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue. A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli. Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species. The apo-domain can be lipoylated in vitro with partially purified E. coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase). Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains.

Acetyltransferases↗
PubMed1974

Differential effects of 2-oxo acids on pyruvate utilization and fatty acid synthesis in rat brain.

1. The effects of 2-oxo-4-methylpentanoate, 2-oxo-3-methylbutanoate and 2-oxo-3-methylpentanoate on the activity of pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), acetyl-CoA carboxylase, (EC 6.4.1.2) and fatty acid synthetase derived from the brains of 14-day-old rats were investigated. 2. The pyruvate dehydrogenase enzyme activity was competitively inhibited by 2-oxo-3-methylbutanoate with respect to pyruvate with a K(i) of 2.04mm but was unaffected by 2-oxo-4-methylpentanoate or 2-oxo-3-methylpentanoate. 3. The citrate synthase activity was inhibited competitively (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (K(i)~7.2mm) and 2-oxo-3-methylbutanoate (K(i)~14.9mm) but not by 2-oxo-3-methylpentanoate. 4. The acetyl-CoA carboxylase activity was not inhibited significantly by any of the 2-oxo acids investigated. 5. The fatty acid synthetase activity was competitively inhibited (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (K(i)~930mum) and 2-oxo-3-methylpentanoate (K(i)~3.45mm) but not by 2-oxo-3-methylbutanoate. 6. Preliminary experiments indicate that 2-oxo-4-methylpentanoate and 2-oxo-3-phenylpropionate (phenylpyruvate) significantly inhibit the ability of intact brain mitochondria from 14-day-old rats to oxidize pyruvate. 7. The results are discussed with reference to phenylketonuria and maple-syrup-urine disease. A biochemical mechanism is proposed to explain the characteristics of these diseases.

Acetyl-CoA Carboxylase↗
PubMed2006

The structure of MbtI from Mycobacterium tuberculosis, the first enzyme in the biosynthesis of the siderophore mycobactin, reveals it to be a salicylate synthase.

The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. Mycobacterium tuberculosis acquires iron exclusively via the siderophore mycobactin T, the biosynthesis of which depends on the production of salicylate from chorismate. Salicylate production in other bacteria is either a two-step process involving an isochorismate synthase (chorismate isomerase) and a pyruvate lyase, as observed for Pseudomonas aeruginosa, or a single-step conversion catalyzed by a salicylate synthase, as with Yersinia enterocolitica. Here we present the structure of the enzyme MbtI (Rv2386c) from M. tuberculosis, solved by multiwavelength anomalous diffraction at a resolution of 1.8 A, and biochemical evidence that it is the salicylate synthase necessary for mycobactin biosynthesis. The enzyme is critically dependent on Mg2+ for activity and produces salicylate via an isochorismate intermediate. MbtI is structurally similar to salicylate synthase (Irp9) from Y. enterocolitica and the large subunit of anthranilate synthase (TrpE) and shares the overall architecture of other chorismate-utilizing enzymes, such as the related aminodeoxychorismate synthase PabB. Like Irp9, but unlike TrpE or PabB, MbtI is neither regulated by nor structurally stabilized by bound tryptophan. The structure of MbtI is the starting point for the design of inhibitors of siderophore biosynthesis, which may make useful lead compounds for the production of new antituberculosis drugs, given the strong dependence of pathogenesis on iron acquisition in M. tuberculosis.

Amino Acid Sequence↗
PubMedDate not supplied

[Isolation, purification and properties of acetolactate synthase from cultured Lactococcus lactis].

Acetolactate synthase catalyzing the synthesis of alpha-acetolactate was isolated from lactic acid bacteria Lactococcus lactis subsp. lactis biovar. diacetylactis 4 and purified. Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the Km for pyruvate was 70 mM. The curve relating the dependence of enzyme activity on pyruvate concentration had a sigmoid shape. The enzyme activity persisted for 24 h in the presence of stabilizers, pyruvate, and thiamine pyrophosphate. Acetolactate synthase had the pH optimums of 5.8 and 6.5-7.0 in acetate and phosphate buffers, respectively. The temperature optimum for this enzyme was 38-40 degrees C at pH 6.5. The molecular weight of acetolactate synthase was 150 kDa. In Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consisted of three identical subunits with a molecular weight of 55 kDa.

Acetolactate Synthase↗
PubMed2001

A metabolic node in action: chorismate-utilizing enzymes in microorganisms.

The shikimate pathway has been described as a metabolic tree with many branches that led to the synthesis of an extensive range of products. This pathway is present only in bacteria, fungi, and plants. While there is only little difference in the sequence of the chemical reactions of the pathway, significant differences exist in terms of organization and regulation. In the main trunk of the shikimate pathway, D-erythrose 4-phosphate and phosphoenolpyruvate are converted via shikimate to chorismate. Chorismate is the common precursor for the biosynthesis of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, but also for other products as diverse as folate cofactors, benzoid and naphthoid coenzymes, phenazines, and siderophores. Five chorismate-utilizing enzymes have been characterized in microorganisms: chorismate mutase, anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and chorismate pyruvate-lyase. In this review these enzymes are discussed in terms of the corresponding gene structures and regulation, nucleotide and protein sequences, protein structures, and reaction mechanisms. The main emphasis is on transcriptional and posttranslational regulatory mechanisms, in view of how a microbial cell exploits its chorismate pool in diverse anabolic pathways. Comparison of the chorismate-utilizing enzymes has shown that some of them share sequence similarity, suggesting divergent evolution and commonality in reaction mechanisms. However, other chorismate-utilizing enzymes are examples of convergent evolution toward similar reaction capabilities.

Anthranilate Synthase↗
PubMed1981

Mechanisms of hormonal regulation of liver metabolism.

The mechanism of actions of glucagon, alpha- and beta-adrenergic agonists, vasopressin and angiotensin II in the liver proposed in this article are summarized in Fig. 8. The actions of glucagon and beta-adrenergic agonists in liver can be entirely ascribed to their interaction with specific plasma membrane receptors which activate adenylate cyclase leading to the intracellular accumulation of cAMP and activation of cAMP-dependent protein kinase. This enzyme phosphorylates phosphorylase b kinase, glycogen synthase, L-type pyruvate kinase, and other liver proteins resulting in alterations in their activities which can account for several of the known hepatic responses to glucagon. There is no clear evidence that Ca2+ ions are involved in the hepatic actions of this hormone. Glucocorticoids, but not thyroid hormones, are required for normal responsiveness of the liver to glucagon. The steroids do not modify cAMP accumulation or cAMP-dependent protein kinase activation, but may act by modulating the action of the kinase on its substrates. Glucocorticoids and thyroid hormones decrease beta-adrenergic responses in the liver apparently by decreasing the number of beta-receptors. Insulin inhibits the actions of physiological concentrations of glucagon by decreasing cAMP accumulation: its mechanism of action is unknown. The actions of alpha-adrenergic agonists, vasopressin and angiotensin II on the liver resemble those of glucagon, but do not involve accumulation of cAMP or activation of cAMP-dependent protein kinase. These agents appear to act by increasing cytosolic Ca2+ thus altering the activities of Ca2+-sensitive enzymes such as phosphorylase b kinase and calmodulin-dependent glycogen synthase kinase. Their receptors appear to be located exclusively on the plasma membrane and a major mechanism by which they raise cytosolic Ca2+ is by inducing the release of this cation from mitochondria. These considerations imply the existence of an intracellular messenger(s) for these agents which is generated at the plasma membrane in response to receptor activation and exerts effects on mitochondria or perhaps other intracellular structures. Glucocorticoids and thyroid hormones increase alpha-adrenergic responses in the liver apparently by increasing the number of alpha-receptors. Insulin inhibits the responses of the liver to alpha-agonists, but not to vasopressin or angiotensin II.

Adrenergic alpha-Agonists↗
PubMed1991

The effect of dietary vitamin E and beta-carotene on oxidation processes in the rat testis.

The effects of dietary vitamin E and beta-carotene were studied on enzymes involved in arachidonic acid metabolism and other related enzymes in the rat testis. Groups of rats were fed various soybean oil-based semi purified diets. Group 1 was fed a vitamin E-supplemented diet (+E - beta); Group 2 was fed a beta-carotene-supplemented diet (-E + beta); Group 3, the control group (-E - beta) was fed a vitamin E-deficient diet; and Group 4, the standard diet group (S), was fed vitamin E plus beta-carotene-standard diet. Soybean oxidized oil was added to the three diet groups - (+E - beta), (- E + beta) and (- E - beta), whereas the diet of S group contained non-oxidized oil. After 8 weeks rats were killed, blood and testis samples were collected for biochemical determinations. Vitamin E deficiency caused significant increase in testis thiobarbituric acid value and activities of testis NADPH oxidase, testis 15-lipoxygenase and in plasma pyruvate kinase. In contrast, significant decreases were observed in activity of testis prostaglandin synthetase, compared with antioxidant-supplemented diet groups. We also found a significant increase in 15-lipoxygenase activity in (- E + beta) diet group, compared with (- E - beta) diet group. Fatty acid analysis of testis parenchyma indicated decrease in palmitate (16:0) and arachidonate (20:4(n - 6)), and increase in oleate (18:1(n-6)) linoleate (18:2(n - 6)) and linolenate (18:3(n - 3)), when compared (-E - beta) diet group with vitamin E-supplemented diet groups. The results suggest that dietary vitamin E has a role in both enzymatic and non-enzymatic peroxidation of polyunsaturated fatty acids in the testis.

Animals↗
PubMed1991

Production of a monocot-specific monoclonal antibody against acetohydroxyacid synthase and its use in the purification and characterization of the enzyme.

Acetohydroxyacid synthase [AHAS; acetolactate pyruvate-lyase (carboxylating), EC 4.1.3.18], the first enzyme unique to the biosynthesis of valine, leucine, and isoleucine, is a known target for several different chemical classes of herbicides. Antibodies required for immunological characterization of the enzyme have not been generated by the conventional method of antibody production using purified protein. Monoclonal antibodies were raised against AHAS from corn by using as immunogen a synthetic peptide representing this enzyme. This antibody immunoprecipitated the enzyme activity from corn. On a Western blot, a protein band with a molecular weight of 65,000 was detected in crude extracts of corn. Furthermore, a monoclonal antibody immunoaffinity gel was used to isolate a single protein from crude enzyme preparations that migrated at Mr 65,000 in an SDS/polyacrylamide gel. The molecular weight of this protein band is the molecular weight predicted for a plant AHAS from a cloned gene sequence. These results strongly suggest that the Mr 65,000 protein represents AHAS in corn extracts. Interestingly, the monoclonal antibody specifically recognized the enzyme from monocots and did not crossreact with AHAS from any dicot species tested. Identification of this monoclonal antibody that distinguishes monocot and dicot AHAS is significant because of a very high degree of amino acid conservation (85%) between AHAS proteins from different species.

Acetolactate Synthase↗
PubMed2006

Proteomic analysis of rat hippocampus and frontal cortex after chronic treatment with fluoxetine or putative novel antidepressants: CRF1 and NK1 receptor antagonists.

Chronic administration of antidepressants is required for their efficacy, suggesting the involvement of long-term modifications. As the impact of antidepressant treatment on the brain molecular machinery is not completely understood, we performed a proteomic analysis of rat hippocampus and frontal cortex after chronic treatment with fluoxetine, with an NK1 receptor antagonist, GR205171, and a CRF receptor 1 antagonist, DMP696. After 2D electrophoresis, protein expression levels were compared with both univariate and multivariate statistical analyses and identified by mass spectrometry. All treatments modified levels of actin isoforms, whereas both fluoxetine and GR205171 reduced synapsin II. Fluoxetine treatment increased ERK2 and NP25 and decreased vacuolar ATP synthase. After GR205171 treatment, protein disulphide isomerase A was reduced; dynamin 1 and aldose reductase increased. DMP696 modulated DRP2, pyruvate kinase, LDH and ATP synthase. Although each compound induced a specific pattern of protein modulation, data suggest that antidepressants share the ability of modulating neural plasticity.

Animals↗
PubMed1995

Excessive glucose production, rather than insulin resistance, accounts for hyperglycaemia in recent-onset streptozotocin-diabetic rats.

Glucose production and utilization and activities of key enzymes involved in liver and muscle glucose metabolism were studied in post-absorptive streptozotocin-diabetic rats after 12 h of severe hyperglycaemia (17.5 +/- 0.5 mmol/l) and insulinopenia (5 +/- 1 microU/ml). Basal glucose production was increased: 36.6 +/- 3.0 mg.kg.min-1, vs 24.4 +/- 2.5 in controls (p < 0.05); liver glycogen concentration was decreased by 40% (p < 0.05); liver phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities were increased by 375 and 156%, respectively (p < 0.001 and < 0.01). During a euglycaemic clamp at a plasma insulin level of 200 microU/ml, glucose production was totally suppressed in controls, but persisted at 20% of basal in diabetic rats. In these rats, glucose production was suppressed at a plasma insulin level of 2500 microU/ml. Basal whole body glucose utilization rate, 2-deoxy-1-[3H]-D-glucose ([3H]-2DG) uptake by muscles and muscle glycogen concentrations were similar in both groups, as well as total and active forms of pyruvate dehydrogenase and glycogen synthase activities. During the euglycaemic clamp, the total body glucose utilization rates and [3H]-2DG uptake by muscles were similar in control and diabetic rats at a plasma insulin level of 200 microU/ml, but lower in diabetic rats at a plasma insulin level of 2500 microU/ml. We conclude 1) in recent-onset severely insulinopenic rats, an excessive glucose production via gluconeogenesis prevailed, mainly accounting for the concomitant hyperglycaemia. This excess glucose output cannot be attributed to liver insulin resistance: the gluconeogenic pathway is physiologically less sensitive than glycogenolysis to the inhibition by insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed2006

Mitochondrial matrix phosphoproteome: effect of extra mitochondrial calcium.

Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondrion-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondrial matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca(2+)) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis-mass spectrometry of Pro-Q Diamond staining, while many more Pro-Q Diamond-stained proteins evaded mass spectrometry detection. Time-dependent (32)P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins that were detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase, and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondrial matrix is extensive and dynamic. Ca(2+) has previously been shown to activate various dehydrogenases, promote the generation of reactive oxygen species (ROS), and initiate apoptosis via cytochrome c release. To evaluate the Ca(2+) signaling network, the effects of a Ca(2+) challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca(2+)-induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca(2+) dose-dependent dephosphorylation of MnSOD was associated with an approximately 2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca(2+). These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca(2+) modification of mitochondrial function at the level of MnSOD.

Animals↗