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:2.3.1.21 (CPT)
4,580 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The T7gene-4 protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA-priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initate DNA synthesis on various circular single-stranded DNA templates by a mechanism which involes the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3':5'-phosphodiester bond, starts with pppA-C and terminates predominantly with AMP. When only ATP and CPT are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA-C-C-A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initators, T7 DNA-priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA-priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template-primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2--4 fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from imitation of DNA chains de novo. Incubation of three proteins: T7 DNA-priming protein, T7 DNA polymerase, and T7 DNA-binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with phiX174DNA as template leads to the generation of 'rolling circle-like' structures as visualized in the electron microscope. Single-stranded regions at the tail-circle junction indicate that initations can occur de novo on the displaced complementary strand. This is consistent with a discontinuous mode of 'lagging' strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo.
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PMID:Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication. 32 3

1. The activities of long-chain acyl-CoA synthetase (acid: CoA ligase (AMP-forming), EC 6.2.1.3) and the "outer" carnitine long-chain acyltransferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21) have been estimated in intact brown adipose tissue mitochondria. The assay of both enzymes is based on a coupled reaction in which the intramitochondrial (matrix) CoASH is the final acyl acceptor and the oxidation-reduction state of the flavoproteins in the acyl-CoA dehydrogens pathway is used to determine the intramitochondrial level of acyl-CoA. 2. Using endogenous fatty acids as the substrate, the progress curve of acyl-CoA synthetase activity was in most mitochondrial preparations linear within the first 30 s. When initial rates were measured, the Km value for CoASH (2.4 micron) was lower than previously determined for the acyl-CoA synthetase in brown adipose tissue mitochondria as well as in mitochondria of other tissues. The pH activity curve indicates that the unprotonated form of the fatty acids represents the substrate of acyl-CoA synthetase, i.e. similar to the effect of pH on the binding of fatty acids to bovine serum albumin. 3. Experimental evidence is presented that at temperatures higher than the transition temperature of the acyl-CoA synthetase (i.e. Tt = 19 degrees C), this enzymic reaction is rate-limiting in the sequence of coupled reactions leading to beta-oxidation in the mitochondrial matrix. 4. The initial rate of the long-chain acyl-COA synthetase reaction was estimated to v = 119 +/- 16 nmol . min-1 . mg-1 protein (mean +/- S.D., n = 5) at an optimal concentration of palmitate which exceeds that of rat heart mitochondria by a factor of 10.
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PMID:Long-chain acyl-CoA synthetase and "outer" carnitine long-chain acyltransferase activities of intact brown adipose tissue mitochondria. 69 44

Palmitoyl-CoA synthetase activity in the microsomal fraction of rat liver was measured directly by palmitoyl-CoA production, and indirectly by converting the palmitoyl-CoA into palmitoylcarnitine under optimum conditions. Even in the latter system, palmitoyl-CoA accumulated. The rate of palmitoyl-CoA hydrolysis and the inhibition of palmitoyl-CoA synthetase by palmitoyl-CoA were each estimated to be less than 10% of the maximum rate of palmitoyl-CoA production. The concentration of palmitoyl-CoA present in the assay systems used for measuring palmitate esterification to glycerol phosphate and the activity of palmitoyl-CoA synthetase by using the carnitine-linked determination were measured. These concentrations were not altered by the addition of glycerol phosphate, or of carnitine plus carnitine palmitoyltransferase. The relationship between the activity of palmitoyl-CoA synthetase and the rate of glycerolipid synthesis was investigated. The latter activity was measured by using palmitoyl-CoA generated from palmitate, palmitoyl--AMP or palmitoylcarnitine. It is concluded that, at optimum substrate concentrations, the activity of glycerol phosphate acyltransferase is rate-limiting in the synthesis of phosphatidate by rat liver microsomal fractions. The implications of these results in the measurement of palmitoyl-CoA synthetase and in the control of glycerolipid synthesis are discussed.
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PMID:Palmitate activation and esterification in microsomal fractions of rat liver. 81 35

1. Long-chain acid: CoA ligase (AMP-forming) (trivial name acyl-CoA synthetase; EC 6.2.1.3) is located at the membranes of the endoplasmic reticulum and the outer membrane of the mitochondria. The latter membrane has by far the highest specific activity. 2. GTP-dependent synthesis of acyl-CoA has a very low activity in liver mitochondria (about 5% of the activity measured with ATP). CTP, ITP, UTP and GTP may all provide energy for fatty acid activation in sonicated mitochondria by formation of ATP from endogenous ADP and AMP. 3. In rat liver palmitoyl-CoA: L-carnitine O-palmitoyltransferase (trivial name carnitine palmitoyltransferase; EC 2.3.1.21) is located at the microsomal membranes and in the inner membrane of the mitochondria. Its activity is increased, in both membranes, during fasting and in thyroxine-treated rats. The extramitochondrial carnitine palmitoyltransferase may capture part of the acyl CoA formed at the endoplasmic reticulum as acyl-carnitine, especially during fasting and other metabolic conditions of high fatty acid turnover. This transport form of activated fatty acid can penetrate the inner mitochondrial membrane (the acyl-CoA barrier) where it can be reconverted to acyl-CoA, providing the substrate for beta-oxidation in the inner membrane-matrix compartment. The small part of the mitochondrial carnitine palmitoyltransferase, described to be present at the external surface of the mitochondrial inner membrane, may have the same function in the transport of acyl-CoA formed at the mitochondrial outer membrane. 4. Isolated rat liver mitochondria can oxidize high concentrations of palmitate or oleate in the absence of carnitine. In this case the fatty acids are activated in the inner membrane-matrix compartment of the mitochondria, probably by a medium-chain acyl-CoA synthetase with wide substrate specificity. Because this enzyme is less active in heart and absent in skeletal muscle, these tissues oxidize long-chain fatty acids in an obligatory carnitine-dependent fashion. Also the liver oxidizes long-chain fatty acids in a carnitine-dependent way if lower fatty acid concentrations are used. In this tissue carnitine stimulates specifically the partial oxidation of fatty acids to beta-hydroxybutyrate and acetoacetate. 5. The activities of acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase (trivial name glycerophosphate acyltransferase; EC 2.3.1.15) and carnitine palmitoyltransferase change in opposite directions during fasting. These activity changes, together with the measured kinetic properties of the enzymes in mitochondria and microsomes, allow a switch (relatively) from lipid synthesis to ketogenesis during fasting. This switch may occur at the level of long-chain acyl-CoA both in the endoplasmic reticulum and in the mitochondria.
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PMID:Aspects of long-chain acyl-COA metabolism. 113 97

We have investigated the interaction between hypothalamic ACTH secretagogues and adrenocortical glucocorticoids in rat anterior pituitary tissue using an in vitro perifusion system. Repeated 5 min pulses of 41-residue CRF (CRF-41) or arginine vasopressin (AVP) were applied at 1 h intervals for up to 7 h. Administration of 0.1 microM corticosterone 30 min before and during the 5 min 0.1 nM CRF-41 stimulus at 5 h resulted in a significant inhibition of CRF-41 stimulated ACTH release within 30 min. Inhibition of ACTH release also developed if no CRF-41 stimulus was applied in conjunction with steroid at 5 h. In contrast, if the exposure to corticosterone (0.1 microM, 35 min total duration) was started simultaneously with the application of CRF-41 at 5 h, no inhibition of ACTH release ensued. Similarly, no inhibition of CRF-41-stimulated ACTH release was observed when corticosterone was started simultaneously with a 5 min pulse of cyclic 8-(4-Chlorophenylthio) AMP (8-CPT-cAMP), a cell membrane permeant analog of cAMP. In contrast to CRF-41 and 8-CPT-cAMP, AVP failed to modify glucocorticoid-induced inhibition of AVP- or CRF-41-stimulated ACTH release. Moreover, CRF-41 did not prevent the glucocorticoid-induced inhibition of AVP-stimulated ACTH release. In summary: 1) CRF-41 inactivates early glucocorticoid inhibition of CRF-41-stimulated ACTH secretion, and this is mimicked by a cell membrane permeant analog of cAMP; 2) AVP does not inactivate glucocorticoid-induced inhibition of stimulated ACTH release; 3) the data point to an acute interaction between the cAMP/protein kinase A and glucocorticoid-responsive intracellular pathways. Such differential modulation of feedback inhibition by CRFs may be of functional importance in vivo.
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PMID:Inactivation of early glucocorticoid feedback by corticotropin-releasing factor in vitro. 131 50

Hepatocytes respond to stimulation by glycogenolytic agonists acting via phosphoinositide (PI) breakdown through oscillations of the free cytosolic concentration of Ca2+ ([Ca2+]cyt.). Since the second-messenger repertoire of hepatocytes includes many other factors besides Ca2+, we investigated to what degree the regulation of [Ca2+]cyt. oscillations is integrated into these other signalling systems. [Ca2+]cyt. was recorded in single rat hepatocytes by using the Ca(2+)-indicator fura-2. Parallel stimulation with phenylephrine (an alpha 1-adrenergic agonist of PI breakdown) and glucagon resulted in a synergistic stimulation of [Ca2+]cyt. oscillations. Direct activation of the cyclic-AMP-dependent pathway with several stimuli (forskolin, 8-bromo cyclic AMP, 8-CPT cyclic AMP) mimicked the response to glucagon. In contrast, [Ca2+]cyt. oscillations induced by various combinations of these agonists could be antagonized by the glycogenic hormone insulin. As one of the options in the insulin-signalling network, we tested a diacylglycerol activator of protein kinase C, DiC8. It also acted as an inhibitor of [Ca2+]cyt. oscillations. We investigated how these observations could be reconciled with our previously introduced model of [Ca2+]cyt. oscillations in hepatocytes [Somogyi and Stucki (1991) J. Biol. Chem. 266, 11068-11077]. First of all, the effect of calmodulin inhibitors (calmidazolium and CGS 9343 B), acting at the core of our model on the feedback of Ca2+ on Ins(1,4,5)P3-induced Ca2+ release, was not altered by the new modulators. In addition, all agonists and antagonists could be used interchangeably in combination and introduced no significant change in the oscillatory pattern or spike shape. Since the response was solely limited to frequency modulation, over- or understimulation of the oscillatory system, there is no need to create a new oscillator or to introduce further reaction steps into the core of the model. We conclude that the regulation of [Ca2+]cyt. via the explored second-messenger pathways can be embedded into the oscillatory system as modulation of rate constants already present in this model.
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PMID:Modulation of cytosolic-[Ca2+] oscillations in hepatocytes results from cross-talk among second messengers. The synergism between the alpha 1-adrenergic response, glucagon and cyclic AMP, and their antagonism by insulin and diacylglycerol manifest themselves in the control of the cytosolic-[Ca2+] oscillations. 132 20

8-(4-Chlorophenyl)thio-cyclic AMP (8-CPT-cAMP), extensively used as selective activator of cyclic AMP-dependent protein kinase, has been found to be a potent inhibitor of the cyclic GMP-specific phosphodiesterase (PDE VA). Indeed, 8-CPT-cAMP (IC50 = 0.9 microM) inhibited PDE VA with a potency identical to that of zaprinast. 8-CPT-cAMP was also metabolized by PDE VA at a rate half that of cyclic GMP. The cyclic GMP-inhibited phosphodiesterase (PDE III) (IC50 = 24 microM) and the cyclic AMP-specific phosphodiesterase (PDE IV) (IC50 = 25 microM) were also inhibited by 8-CPT-cAMP. In contrast, most of the other cAMP-derivative studies showed little inhibition of any phosphodiesterase isoenzyme. These observations provide further reasons why the mechanism of the physiological effects of 8-CPT-cAMP should be interpreted with caution.
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PMID:8-(4-Chlorophenyl)thio-cyclic AMP is a potent inhibitor of the cyclic GMP-specific phosphodiesterase (PDE VA). 133 52

1. The effect of the microtubule-disruptive agent, colcemid (N-deacetyl-N-methyl-colchicine), on the water permeability response to vasopressin has been investigated in isolated cortical collecting tubules from the rabbit kidney perfused in vitro. 2. Pretreatment of collecting tubules with colcemid inhibited the increase in water permeability elicited by vasopressin, 50 microU ml-1, in a time- and dose-dependent manner. After 75 min exposure to the drug, inhibition of the response to the hormone averaged 72 +/- 6% (n = 4, P < 0.01) at a colcemid concentration of 7.2 x 10(-5) M. Inhibition was estimated to be half-maximal at a colcemid concentration of 1.9 x 10(-6) M. 3. Colcemid, 2.7 x 10(-7) to 7.2 x 10(-5) M, had no effect on basal water permeability nor on the increase in lumen negative potential difference (PD) induced by the hormone. 4. Lumicolcemid, an isomer of colcemid that does not disrupt microtubules, had no influence on the water permeability response to vasopressin. 5. Pretreatment with colcemid, 2.7 x 10(-5) M, for 45 min inhibited the water permeability response to 8-CPT-cAMP, 1.8 x 10(-5) M, by 38 +/- 4% (n = 5, P < 0.01). 6. When collecting tubules were exposed to colcemid, 5.5 x 10(-5) M, for 45 min after the hydrosmotic response to vasopressin had been established, the drug had no influence on the maintenance of the raised water permeability. 7. The results provide further evidence that cytoplasmic microtubules play a role in the initiation of the hydrosmotic response to vasopressin in the mammalian collecting tubule at a site distal to the generation of cyclic AMP.
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PMID:Effect of colcemid on the water permeability response to vasopressin in isolated perfused rabbit collecting tubules. 133 5

Transcription of the rat serine dehydratase (SDH) gene is induced by glucagon, mediated by the action of cAMP. To identify the nucleotide sequences in the SDH gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5' flanking region of the rat SDH gene fused to the structural sequence encoding the bacterial reporter enzyme, chloramphenicol acetyltransferase (CAT). The transcriptional activities of the fusion genes introduced into the rat hepatoma cell line 7AD-7 were assayed by measuring CAT activity in the cell lysates. Chlorophenylthio-cyclic AMP (CPT-cAMP), a potent protein kinase A activating agent, stimulated the expression of SDH-CAT fusion genes, and these inductions could be enhanced further by the addition of dexamethasone, although the glucocorticoid alone had no effect on CAT activity. Deletion analysis demonstrated that an 80 bp region located approximately 3.5 kb upstream from the transcription initiation site of the rat SDH gene was responsible for stimulation of transcription by CPT-cAMP, whereas the 120 bp region immediately upstream of the cAMP responsive element (CRE)-containing sequences is essential for the enhancement of CPT-cAMP induction by the glucocorticoid.
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PMID:Identification of regions in the rat serine dehydratase gene responsible for regulation by cyclic AMP alone and in the presence of glucocorticoids. 133 28

We investigated the role of energy supplied by long-chain fatty acid oxidation in rat platelet function. Inhibition of the mitochondrial uptake of long-chain fatty acids was achieved by treating rats with 2-tetradecylglycidic acid (TDGA), a potent inhibitor of the overt form of carnitine palmitoyltransferase (CPT-I). The maximum aggregation rate (MAR), CPT-I activity, lactate production, oxygen consumption and adenine nucleotide content of isolated rat platelets were then studied in vitro. 4 h after the in vivo administration of TDGA, the CPT-I activity in saponin-permeabilized platelets was nearly completely inhibited along with a significant reduction in the MAR induced by ADP, thrombin and ionophore A23187. The ATP level, adenylate energy charge (ATP + 1/2 ADP)/(ATP + ADP + AMP) and ATP/ADP ratio in the platelet cytoplasmic pool were also reduced. Platelets from TDGA-treated rats showed lower oxygen consumption rates in both the basal respiratory and oxygen burst states. These results indicate that mitochondrial long-chain fatty acid oxidation coupled to oxidative phosphorylation is an important energy source in rat platelets and is probably involved in the maintenance of platelet function. Enhanced in vitro lactate production in platelets from TDGA-treated rats may have resulted from a compensatory increase in glycolysis which only partly compensated for impaired long-chain fatty acid oxidation.
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PMID:Effects of 2-tetradecylglycidic acid on rat platelet energy metabolism and aggregation. 142 Feb 90


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