Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of the catalytic subunit of bovine cardiac muscle cAMP-dependent protein kinase with N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the most potent and selective inhibitor toward cyclic nucleotide-dependent protein kinases in the series of isoquinolinesulfonamide derivatives, was studied. The addition of H-8 protected the catalytic subunit of the enzyme in a dose-dependent manner from irreversible inactivation by the ATP analogue p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). The inactivation followed pseudo-first order kinetics and H-8 reduced the steady state constant of inactivation (Ki) without any effect on the first order rate constant (K3). The quantitative binding of H-8 to the enzyme was measured under conditions of thermodynamic equilibrium using a gel filtration method. The catalytic subunit bound approximately 1 mol of drug/mol of protein with apparent half-maximal binding at 1.0 microM drug, whereas the enzyme irreversibly modified by FSBA did not bind the drug, confirming that the enzyme has no site for H-8 in the catalytic subunit other than the active site. The binding studies also showed that H-8 does not require divalent cations such as Mg2+ to bind to the catalytic subunit of the protein kinase. The binding of H-8 to the active site was characterized using FSBA and other affinity labeling reagents which have been postulated to modify residues at or near the active site of the catalytic subunit. H-8 protected the enzyme against inactivation by FSBA and Cibacron Blue F3GA but did not afford any protection against the covalent modification of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), suggesting that the binding site of H-8 does not involve the gamma-subsite of the ATP binding site in the catalytic subunit, since DTNB and NBD-Cl are thought to modify the residues complementary to gamma-phosphate of the ATP molecules.
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PMID:Specific binding of a novel compound, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) to the active site of cAMP-dependent protein kinase. 357 96

The involvement of a purinergic system in the mechanisms of ATP- and electrically induced long-term potentiation (LTP) has been investigated in mouse hippocampal slices. Extracellular ATP (500 nM) and its slowly hydrolyzable analogue adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S; 2.5 microM) amplified permanently the magnitude of the population spike. This effect was antagonized by adenylimidodiphosphate (AMPPNP), a non-hydrolyzable analogue of ATP. AMPPNP, other ATP analogues [2-methylthioadenosine triphosphate (2-MeSATP) and alpha, beta-methyleneadenosine 5'-triphosphate (alpha, beta-methyleneATP)], or a purinergic receptor antagonist (Cibacron Blue 3G) tested in the concentration range of 3-40 microM did not exert agonistic activity similar to that of ATP or ATP-gamma-S, suggesting that ATP hydrolysis is required to exert this effect. All the tested nonhydrolyzable analogues reduced or prevented the establishment of stable, nondecremental LTP without blocking the short-lasting increase in the magnitude of the population spike immediately after electrical stimulation (short-term potentiation). These results indicate that ATP released by high-frequency stimulation contributes to the maintenance of stable LTP. The underlying mechanism operating in this process may involve a new type of ATP receptors or hydrolysis by ecto-ATPase. However, the findings that ATP-gamma-S is less potent than ATP and that other ATP analogues known to act as agonists of purinergic receptors did not induce LTP but rather inhibited its maintenance are more consistent with the possibility that ecto-protein kinase, using extracellular ATP as a cosubstrate, plays a role in mechanisms underlying synaptic plasticity.
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PMID:On the role of extracellular ATP in the induction of long-term potentiation in the hippocampus. 793 28

Supporting cells in the mammalian cochlea have recently received attention as potential targets of neurotransmitters, neuromodulators, and neurohumoral agents. Calcium homeostasis in Deiters' and Hensen's cells, for example, is regulated by ATP and nitric oxide. We studied the intracellular calcium concentration [Ca2+]i in isolated pillar cells of the guinea pig cochlea in response to extracellular ATP and nitric oxide using the fluorescent indicator fluo-3. [Ca2+]i increased rapidly and significantly throughout the pillar cell in response to a bolus of ATP or 2-methylthio ATP while alpha,beta-methylene ATP was ineffective. The response to ATP was inhibited by suramin and Cibacron Blue but not by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid. This pharmacological profile is consistent with a [Ca2+]i increase largely mediated by P2Y receptors. In Ca2+-free medium supplemented with EGTA, the response to extracellularATP was reduced by 33%, suggesting a contribution of calcium influx to the overall effect. The ATP-induced increase of [Ca2+] was attenuated by NO donors (sodium nitroprusside or diethylamine NONOate), and this attenuation was reversed by KT5823, an antagonist to protein kinase G. The results indicate the involvement of purinergic mechanisms and the nitric oxide/cyclic GMP/protein kinase G pathway in the regulation of [Ca2+]i in cochlear pillar cells.
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PMID:ATP and nitric oxide modulate intracellular calcium in isolated pillar cells of the guinea pig cochlea. 1183 12

Infection of target cells by alphaherpesviruses leads to extensive modulation of host cell gene expression. To gain detailed information on the molecular pathways affected by infection of Madin-Darby bovine kidney (MDBK) cells with PrV, transcript analysis was combined with a stable isotope-based quantitative proteomic approach (SILAC). Four hours after infection cells were harvested and processed in parallel either for transcript analysis, for subcellular fractionation into nuclei and cytosol, for extraction of phosphoproteins, or for affinity extraction with Heparin Sepharose and Cibacron Blue F3G-A-Sepharose. All fractions were further analysed by large format two-dimensional gel electrophoresis in different pH-ranges to maximize the number of proteins to be identified and quantified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Cell fractionation was quick and easy to perform but in comparison to affinity fractionation yielded lower numbers of identified and quantified proteins. After infection with PrV, only two of the 55 proteins with significantly modulated protein levels showed significant changes in transcript levels, indicating that posttranslational modifications may play a major role in the cellular response to PrV infection. Application of isotope labelling to cell cultures infected with wild-type PrV-Ka and a US3 protein kinase negative mutant allowed to monitor pUS3-dependent changes in the expression levels of viral proteins pUL29, pUL39 and pUL42.
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PMID:Gene expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques. 2023 42