Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biospecific affinity chromatography has been used to purify specific cyclic AMP and cyclic GMP receptor proteins. Several variables are important for successful purification of the cyclic AMP receptor protein, the most critical being the length of the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to agarose specifically retains the cyclic AMP receptor protein by interaction with the immobilized nucleotide. Binding of the cyclic AMP receptor subunit of cyclic AMP-dependent protein kinase to the immobilized nucleotide results in dissociation of the catalytic protein phosphokinase subunit which is not retained. The retained cyclic AMP receptor protein is subsequently eluted by cyclic AMP. Homogeneous cyclic AMP receptor protein prepared from rabbit skeletal muscle by affinity chromatography has been characterized. The molecular weight of the native protein as determined by analytical ultracentrifugation and polyacrylamide gel electrophoresis at varying acrylamide concentrations is 76 800 and 82 000, respectively. The protein is asymmetric with frictional and axial ratios of 1.64 and 12. SDS and urea polyacrylamide gel electrophoresis indicate that the native cyclic AMP receptor is composed of two identical subunits of 42 700 molecular weight. The native protein dimer binds 2 moles of cyclic AMP per mole of protein and is active in suppressing activity of isolated catalytic subunits of cyclic AMP-dependent protein kinase. Cyclic GMP receptor protein from bovine lung has been purified using the same affinity chromatography media. Since cyclic nucleotide binding to cyclic GMP-dependent protein kinase does not result in dissociation of regulatory receptor and catalytic phosphotransferase subunits, the cyclic GMP-dependent protein kinase holoenzyme is retained on the column and can be subsequently specifically eluted with cyclic GMP.
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PMID:The use of affinity chromatography in purification of cyclic nucleotide receptor proteins. 18 11

In healthy humans glucagon infusion resulted in a significant increase in blood sugar and in plasma cyclic AMP. No discernible hemodynamic effects were found. Isoproterenol infusion on a mole per mole basis in the same subjects induced a significant, although less pronounced rise in plasma cyclic AMP, heart rate, and a fall in diastolic blood pressure but had no effect on blood sugar. Propranolol administration abolished the hemodynamic effects of isoproterenol and significantly decreased the response of plasma cyclic AMP; the same blocking dosage had little effect on plasma cyclic AMP changes induced by glucagon wheras the response in blood sugar was significantly reduced. These data in vivo are compatible with the in vitro demonstration of separate receptors for glucagon and isoproterenol.
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PMID:Effects of beta-adrenergic blockade on plasma cyclic AMP and blood sugar responses to glucagon and isoproterenol in man. 18 46

1. Cyclic nucleotide levels and compound action potential magnitudes were measured in frog sciatic nerves following exposure to carbachol, isoprenaline and cyclic nucleotide related substances. 2. The resting cyclic AMP level was 2-4 p-mole/mg protein and the cyclic GMP level was 0-27 p-mole/mg protein in desheathed nerves. 3. Isoprenaline (100 micrometer) caused a twofold increase in cyclic AMP without affecting cyclic GMP levels. Carbachol (100 micrometer) caused a twofold increase in cyclic GMP without affecting cyclic AMP levels. 4. The phosphodiesterase inhibitor theophylline (5 mM) augmented both cyclic AMP and cyclic GMP. 5. The magnitude of the resting or compound action potential was not affected by isoprenaline, carbachol, or phosphodiesterase inhibitors. 6. The cyclic nucleotides and their butyryl derivatives did not affect the magnitude of the resting or compound action potential, either when applied alone or in the presence of a phosphodiesterase inhibitor. 7. In contrast to sympatic tissue we conclude that hormone mediated cyclic nucleotide metabolism in peripheral nerve is unrelated to control of axonal excitability.
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PMID:Modulation of cyclic nucleotide levels in peripheral nerve without effect on resting or compound action potentials. 19 34

The reaction of the phosphate residue transfer catalysed by histone kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) was studied. The phosphotransferase reaction was shown to obey the mechanism of ping-pong bi-bi type. After incubation of the catalytic subunit of histone kinase with [gamma-32P]ATP the incorporation of one mole of [32P]phosphage per mole of protein was observed. The tryptic [32P]phosphohistidine-containing peptide was isolated and its N-terminus and amino acid composition were determined. The 2',3'-dialdehyde derivative of ATP (oATP) was used as the affinity label for the catalytic subunit of cyclic-AMP-dependent histone kinase. The inhibitor formed an alidmine bond with epsilon-amino group of the lysine residue of the active site and was irreversibly bound to the enzyme after reduction by sodium borohydride with concurrent irreversible inactivation of the enzyme. After inactivation, about one mole of 14C-labelled inhibitor was incorporated per mole of the enzyme. ATP effectively protected the catalytic subunit of histone kinase against inactivation by oATP. Tryptic digestion of the enzyme-inhibitor complex led to the isolation of the 14C-labelled peptide of the active site of histone kinase. Basing on these results, the role of histidine and lysine residues in the active site of the catalytic subunit of histone kinase was suggested.
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PMID:Studies on the mechanism of action of histone kinase dependent on adenosine 3':5'-monophosphate. Evidence for involvement of histidine and lysine residues in the phosphotransferase reaction. 20 63

A human skeletal actin.tropomyosin.troponin complex was phosphorylated in the presence of [gamma-32 P]ATP, Mg2+, adenosine 3':5'-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 microM cyclic AMP. In the presence of 10(-7) M Ca2+ and protein kinase 0.1 mole of [32P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [32P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca2+, 5.10(-5) M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [32P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [32P]phosphate. Increasing the Ca2+ concentration did not significantly decrease the [32P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstitute human skeletal actomyosin made with the [32P] phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca2+.
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PMID:Phosphorylation of an actin.tropomyosin.troponin complex from human skeletal muscle. 20 9

The catalytic groups, involved in aminoacyl-tRNA formation remain unknown. The isolation and identification of an active covalent complex between the enzyme and substrate is an essential step in understanding the reaction mechanism. We identified and isolated the covalent complex of tryptophanyl-tRNA synthetase (EC 6.1.1.2) and tryptophane which was able to aminoacylate the tRNATrp in the absence of ATP. In beef pancreas tryptophanyl-tRNA synthetase preparations, isolated by the previously described method, a tightly bound tryptophan was revealed which could not be removed by charcoal treatment, by gel-filtration and by replacement with the excess of typtamine, a competitive inhibitor of tryptophane. This tightly bound tryptophane is able to exchange rapidly and specifically with radioactive tryptophane allowing to obtain [14C]tryptophane-tryptophanyl-tRNA synthetase complex. After the reaction of this complex with NH2OH at neutral pH tryptophanyl hydroxamate is formed proving the activated state of the tryptophane in the initial complex with the enzyme. No nucleotide impurites were noticed in the enzyme preparation; the complex is stable at denaturation. A conclusion is made that the tryptophanyl-tRNA synthetase isolated by our method is a tryptophanyl-enzyme. The tryptophanyl residue could be specifically transferred to tRNATrp in the absence of other substrates of the reaction, the efficiency of the transfer does not exceed 25%. The content of the covalently bound tryptophane never exceeds 1 mole per mole of the dimeric enzyme. The total content of tryptophane in the forms of tryptophanyl-enzyme and tryptophanyl adenylate enzyme complex equals 2 moles per mole of the enzyme. The tryptophanyl-enzyme is destroyed during incubation with AMP or with pyrophosphate. The role of the tryptophanyl-enzyme as a possible intermediate in the course of aminoacylation of tRNATrp is discussed.
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PMID:[Tryptophanyl tRNA synthetase: isolation and characteristics of the tryptophanyl-enzyme]. 20 77

Inhibitor-1 is a protein which inhibits phosphorylase phosphatase only when it has been phosphorylated by cyclic-AMP-dependent protein kinase [Huang, F. L. and Glinsmann, W. H. (1976) Eur. J. Biochem. 70, 419--426]. Inhibitor-1 was purified by a heat treatment at 90 degrees C, precipitation with ammonium sulphate, chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and finally rechromatography of the phosphorylated protein on DEAE-cellulose, The protein was purified 4000-fold and 1.5 mg per 1000 g muscle was obtained in seven days corresponding to an overall yield of 15-20%. The purified protein was in a state approaching homogeneity as judged by the criteria of polyacrylamide-gel electrophoresis and ultracentrifugal analysis. The concentration of inhibitor-1 in vivo was calculated to be 1.5 micron, which is at least as high as the concentration of phosphorylase phosphatase. The amino acid composition of inhibitor-1 showed several unusual features. Glutamic acid and proline accounted for nearly one third of the residues, tyrosine, tryptophan and cysteine were absent, and the content of aromatic amino acids was very low. The molecular weight measured by sedimentation equilibrium centrifugation was 19200 and by amino acid analysis was 20800. These values were lower than the mol. wt 26000 determined empirically by gel electrophoresis in the presence of sodium dodecyl sulphate, and much lower than the apparent molecular weight of 60000 estimated by gel filtration on Sephadex G-100. The gel filtration behaviour, stability to heating at 100 degrees C and amino acid composition suggest that inhibitor-1 may possess little ordered structure. The phosphorylated from of inhibitor-1 contained close to one molecule of covalently bound phosphate per mole of protein, which is consistent with the previous finding of a unique decapeptide sequence at the site of phosphorylation, Ile-Arg-Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Thr- [Cohen, P., Rylatt, D. B. and Nimmo, G. A. (1977) FEBS Lett. 76, 182-186].the phosphorylated form of inhibitor-1 inhibited phosphorylase phosphatase activity (0.02U) by 50% at a concentration of only 7.0 nM in the standard assay, but the phosphorylated decapeptide was 1000-2000 times less effective as an inhibitor.
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PMID:The regulation of glycogen metabolism. Purification and characterisation of protein phosphatase inhibitor-1 from rabbit skeletal muscle. 20 44

1. The influence of adenosine on the release of cyclic adenosine 3',5'-monophosphate (cyclic AMP) from the heart was examined in twenty-two anaesthetized intact dogs. The animals were pre-treated with propranolol and vagotomized. The rate of nucleotide release from the left ventricle was determined as the product of the left ventricular myocardial plasma flow and the coronary veno-arterial difference in plasma nucleotide concentration. 2. Adenosine was infused into the left ventricle during three successive 15 min periods at rates of 25, 50 and 100 n-mole/kg. min, respectively. The mean blood pressure in the ascending aorta was prevented from falling by inflating a balloon placed into the thoracic aorta. The measurements were performed before the infusion and at the 10th min of each infusion period. The values given are means +/- S.E. 3. During the 45 min infusion of adenosine at increasing rate, the cyclic AMP concentration in arterial plasma increased from 7.0 +/- 0.3 to 14.0 +/- 0.9 p-mole/ml. The nucleotide was released from the left ventricle at rates increasing from 48.2 +/- 7.1 to 206.5 +/- 56.2 p-mole/100 g. min while the left ventricular myocardial blood flow increased from 127 +/- 6 to 399 +/- 33 ml./100 g.min. The oxygen consumption of the left ventricle was not modified. 4. When adenosine was infused at a rate of 100 n-mol/kg.min, the thorax was opened and the apex of the heart and the left atrial appendage were removed for nucleotide assay. The cardiac cyclic AMP concentration did not differ from that observed in control dogs. 5. The results suggest that cyclic AMP is likely to be involved in the membrane and cellular events underlying the relaxant effect of adenosine on the coronary smooth muscle. The lack of change in cardiac cyclic AMP concentration, as determined by whole tissue extractions, is consistent with a study by others showing that, under normoxic conditions, cyclic AMP is released from a small, compartmentalized fraction of the cyclic AMP content of the heart. The elevation of the plasma nucleotide concentration could result from adenosine effects on various cell systems or organs, in addition to the observed release from the heart.
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PMID:Adenosine-induced release of cyclic adenosine 3',5'-monophosphate from the left ventricle in the anaesthetized intact dog. 20 81

Plasma cyclic AMP content was determined without being extracted, using binding protein obtained from rat liver. EDTA was suitable as an anticoagulant for cyclic AMP estimation. Cyclic AMP further added to EDTA plasma was able to be estimated. The estimated values by plasma dilution were almost the same as the expected values. It was thought that the direct assay was useful for determination of plasma cyclic AMP. Isoproterenol (50 microgram/kg, iv) produced an increase of plasma cyclic AMP level accompanied with a decrease of blood pressure and an increase of heart rate in anesthetized dogs. Cyclic AMP level of peripheral venous plasma was 18.6 +/- 1.32 p mole/ml in human (N=25), 21.6 +/- 3.04 P mole/ml in dogs (N=7) and 50.6 +/- 4.59 p mole/ml in rabbit (N=9). Plasma cyclic AMP level of rabbit was higher than those of human and dog.
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PMID:A rapid, simple determination of plasma cyclic AMP. 20 99

Bovine liver fructose 1,6-bisphosphatase bound 4 mol of its allosteric inhibitor AMP per mole of enzyme with half-saturation at 17 mumol/l AMP. The presence of a mixture of positive and negative cooperativity in the binding of AMP to the enzyme was suggested by several procedures for analyzing binding data. In particular, calculation of the intrinsic binding constants for AMP yielded the relationships: K1' less than K2' greater than K3' less than K4', indicating mixed cooperativity.
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PMID:Binding of adenosine 5'-monophosphate to bovine liver fructose 1,6-bisphosphatase. 22 77


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