Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The unfolding of the recombinant regulatory subunit of cAMP-dependent protein kinase I was followed by monitoring the intrinsic protein fluorescence. Unfolding proceeds in at least two stages. First, the quenching of fluorescence due to cAMP binding is abolished at relatively low levels of urea (less than 2 M) and is observed as an increase in intensity at 340 nm. The high-affinity binding of cAMP is retained in 3 M urea even though the quenching is lost. The second stage of unfolding, presumably representing unfolding of the polypeptide chain, is seen as a shift in lambda max from 340 to 353 nm. The midpoint concentration, Cm, for this process is 5.0 M. Cyclic AMP binding activity is lost at a half-maximal urea concentration of 3.5 M and precedes the shift in lambda max. Unfolding of the protein in the presence of urea was fully reversible; furthermore, the presence of excess levels of cAMP stabilized the regulatory subunit. A free energy value (delta GDH2O) of 7.1 +/- 0.2 kcal/mol was calculated for the native form of the protein when denaturation was induced with either urea or guanidine hydrochloride. Iodide quenching of tryptophan fluorescence was used to elucidate the number of tryptophan residues accessible during various stages of the unfolding process. In the native cAMP-bound form of the regulatory subunit, only one of the three tryptophans in the regulatory subunit is quenched by iodide while more than two tryptophans can be quenched with iodide in the presence of 3 M urea.
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PMID:Unfolding of the regulatory subunit of cAMP-dependent protein kinase I. 184 84

The mechanical and biochemical responses of the canine trachealis to SK&F 94836 [2-cyano-1-methyl-3-[4-(4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazine-3-yl)phenyl]guanidine], a selective inhibitor (ki = 1-3 microM) of the low km cyclic AMP (cAMP) phosphodiesterase, were assessed. Time course studies indicated that SK&F 94836-induced relaxation of trachealis strips contracted with 0.1 microM methacholine was accompanied by an activation of cAMP-dependent protein kinase (cAMP-PK). In subsequent experiments, trachealis strips were contracted with three concentrations of methacholine (0.1, 1.0 or 3.0 microM) or two concentrations of histamine (10 or 300 microM) before being relaxed by the cumulative addition of SK&F 94836. The relaxant response to SK&F 94836 (EC50 = 1-10 microM) decreased progressively as tissues were contracted with higher concentrations of methacholine. In parallel with its inhibitory effect on SK&F 94836-induced relaxation, methacholine suppressed the ability of SK&F 94836 to activate cAMP-PK. Interestingly, the inhibition of cAMP-PK activity was not accompanied by a significant inhibition of SK&F 94836-stimulated cAMP accumulation. Unlike the results with methacholine, the concentration of histamine used to contract tissues had no effect on SK&F 94836-induced relaxation or cAMP-PK activation. To determine the effect of SK&F 94836 on the mechanical and biochemical responses to the beta adrenoceptor agonist isoproterenol, tissues were first contracted with 3.0 microM methacholine and then incubated with 0, 0.3, 3.0 or 30 microM SK&F 94836 before being relaxed by the cumulative addition of isoproterenol. In these experiments, SK&F 94836 potentiated isoproterenol-induced relaxation, cAMP accumulation and cAMP-PK activation in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of the low km cyclic AMP phosphodiesterase in intact canine trachealis by SK&F 94836: mechanical and biochemical responses. 284 31

A mutant LLC-PK1 cell line, M18, was isolated after a single treatment of the parent culture with N-methyl-N'-nitro-N-nitroso-guanidine. In contrast to LLC-PK1 cells, the mutant did not exhibit production of urokinase-type plasminogen activator (uPA) in response to the hormones calcitonin and vasopressin, but produced the expected levels of uPA upon stimulation by the receptor-independent adenylate cyclase activators forskolin and cholera toxin, as well as by the phosphodiesterase inhibitor isobutylmethylxanthine and the 8-bromo analogue of adenosine cyclic monophosphate, Br8cAMP. The patterns of activation of cAMP-dependent protein kinase were identical to those of uPA induction: calcitonin and vasopressin were without effect, but the response to all other agents was normal. In similar fashion, mutant cell homogenates displayed normal activation of adenylate cyclase upon treatment with sodium fluoride, forskolin, or the non-hydrolyzable GTP analogue guanosine 5'-[beta, gamma-imino]triphosphate, but were unresponsive to calcitonin or vasopressin. The ability of M18 cells to bind radioactively labelled calcitonin and vasopressin was measured. The mutant possessed less than 4% of the normal levels of the receptor binding activity for both hormones. Somatic cell hybrids formed between M18 and LLC-PK1 cells were found to retain normal hormone binding activity and responsiveness to hormones, indicating that the defect in M18 cells was recessive. M18 was concluded most probably to contain a single mutation impairing the function of two distinct polypeptide hormone receptors.
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PMID:Isolation of a mutant LLC-PK1 cell line defective in hormonal responsiveness. A pleiotropic lesion in receptor function. 302 58

The primary structure of phenylalanine hydroxylase purified from rat liver was investigated with high speed gel filtration chromatography, cyanogen bromide cleavage and end group analyses of polypeptides derived from the enzyme. On gel filtration in the presence of 6M guanidine hydrochloride, the enzyme gave a single peak corresponding to a molecular weight of 52,000. In the same system the enzyme that had been cleaved with cyanogen bromide gave two peptides (CB1, Mr = 32,800 and CB2, Mr = 20,400). Sequence studies showed that the alignment of these two peptides was CB1 - CB2. Furthermore, in experiments using 32P phosphorylated enzyme, the site of phosphorylation by cAMP-dependent protein kinase was found to be located on the CB1 peptide. The NH2-terminus of this enzyme, which was found to be blocked, was shown to be N-acetylalanine. By both carboxypeptidase A digestion and hydrazinolysis, the carboxyl terminus was identified as serine. These data indicate that the phenylalanine hydroxylase molecule from rat liver is composed of subunits which are homogenous or, at least, very similar in their primary structure.
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PMID:Studies on the primary structure of rat liver phenylalanine hydroxylase. 397 94

The complete amino acid sequence of the catalytic subunit (gamma subunit) of rabbit skeletal muscle phosphorylase b kinase was determined. The gamma subunit was purified by gel filtration in acidic 8 M urea after reduction and S-carboxymethylation in 7 M guanidine hydrochloride. Cleavage of the gamma subunit at arginyl bonds gave a complete set of nonoverlapping peptides. Overlapping peptides were obtained by cleavage at methionyl, tryptophanyl, or glutamyl bonds and by selected subdigestion of two large peptides obtained by cleavage at methionyl bonds. Sequence analysis established that the protein contains 386 residues corresponding to a molecular weight (Mr) of 44673. Comparison of the gamma subunit with the catalytic subunit of bovine cAMP-dependent protein kinase and with tyrosine-specific kinases of viral origin revealed a significant degree of sequence identity among all of these proteins. These data suggest that calcium-dependent protein kinases may share a common ancestral gene and a common structural basis for catalytic function with a wide variety of other protein kinases which respond to different signals and control quite different processes.
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PMID:Homology of the gamma subunit of phosphorylase b kinase with cAMP-dependent protein kinase. 654 4

Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.
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PMID:Domain-structure analysis of recombinant rat hormone-sensitive lipase. 891 75

The bisphosphatase activity of the hepatic bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is repressed by its kinase domain, and regulated by cAMP-dependent protein kinase (PKA)-catalysed phosphorylation. In the present study, the mechanism by which the bisphosphatase activity is repressed by the kinase domain and regulated by phosphorylation was investigated. We found that truncation of the C-terminus of the enzyme by 25, but not 20, amino acids dramatically enhanced the catalytic rate of the bisphosphatase, abrogated the inhibition by the kinase domain, and eliminated the effect of PKA-mediated phosphorylation on activity. In addition, mutation of His444-Arg-Glu-Arg to Ala-Ala-Glu-Ala had similar effects as the deletion. Moreover, the mutations also significantly affected the phosphorylation-mediated regulation of the kinase activity of the enzyme. Furthermore, the mutations altered the pH-dependence of the bisphosphatase, and the mutant bisphosphatases were more sensitive to modification by diethyl pyrocarbonate and guanidine-induced inactivation than the wild-type enzyme. Taken together, these results demonstrate that the sequence His444-Arg-Glu-Arg plays a critical role in repression of the bisphosphatase activity by both the N-terminal kinase domain and the C-terminal tail itself. These results also explain the activation of the bisphosphatase activity by PKA-catalysed phosphorylation, by suggesting that phosphorylation may relieve the inhibitory effect of the kinase domain that is mediated by the three basic residues in this sequence.
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PMID:Involvement of the chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444-Arg-Glu-Arg in modulation of the bisphosphatase activity by its kinase domain. 1143 2

Fluorescence spectroscopy was used to differentiate between different states of acrylodan-labeled cAMP-dependent protein kinase catalytic subunits in urea, guanidine hydrochloride and 3-(N-morpholino)propanesulfonic acid solutions, by measuring changes in the emission spectrum of the protein-coupled dye, which is very sensitive to its microenvironment. Decomposition of the observed fluorescence spectra by a parameterized log-normal distribution function allowed the resolution of overlapping spectral bands and revealed the formation of three distinct protein states, denominated as native, denatured and unfolded structures. At low denaturant concentrations the formation of the denatured form from the native protein was observed, and this process was characterized by a blue-shift of the fluorescence spectrum of acrylodan, indicating that the dye was transferred into some water-deficit hydrophobic environment inside the protein molecule. Therefore, formation of a "dry molten globule" structure could be suggested in state. At high denaturant concentrations a red-shift of the emission spectrum of the protein-coupled probe was observed indicating significant extrusion of the dye molecule into water environment as a result of the unfolding of the protein structure.
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PMID:Different States of Acrylodan-Labeled 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits in Denaturant Solutions. 2760 Nov 74