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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transient cerebral ischemia demonstrates an increase in activated oxygen species in the brain that could lead to eventual neuronal cell death. Neuronal cells respond to oxygen free radicals through the restructuring of the cytoskeleton and membranes, mobilization of calcium and gene expression which play a role in cell injury. Ten min of bilateral carotid artery occlusion resulted in a decrease in calcium/calmodulin dependent protein kinase II (CaM kinase II) phosphorylation and activity detected in the brain immediately following ischemia and was partially restored within 24 h of reperfusion. Pretreatment of animals with an anesthetic dose of pentobarbital (40 mg/kg) resulted in partial protection of inactivation of CaM kinase II following ischemia. CaM kinase II activity was maintained following pretreatment of animals with alpha-phenyl N-tert-butyl nitrone (PBN), which traps oxygen free radicals. Infusion of superoxide dismutase or catalase prior to ischemia, blocked CaM kinase II inactivation. Blockage of calcium uptake with bepridil resulted in a marked protection of CaM kinase II inactivation. In addition, trifluoperazine, a calmodulin antagonist also diminished the inhibition of CaM kinase II phosphorylation in our model. These results suggest that ischemia and reperfusion injury results in the generation of activated oxygen and the mobilization of calcium which inactivate CaM kinase II. These results indicate that changes associated with protein kinase activity in the brain following an ischemic insult may have profound effects upon neurodegeneration and neuronal survival.
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PMID:Role of calcium in inactivation of calcium/calmodulin dependent protein kinase II after cerebral ischemia. 133 39

Experiments were conducted using undifferentiated U937 cells, a human monocytic cell line, to establish an in vitro model to examine the hormonal regulation of the cyclic AMP (cAMP)-specific phosphodiesterase (PDE IV). Standard chromatographic techniques, coupled with the use of inhibitors and activators that are selective for various phosphodiesterase (PDE) isozymes, were used to establish the PDE isozyme profile in supernatant fractions of U937 cells. When PDE activity was assessed using 1 microM [3H]cAMP as a substrate, 70 to 90% of the total U937 cell supernatant activity in the major peak eluting from anion-exchange columns was inhibited by 30 microM rolipram, a selective inhibitor of PDE IV. The remaining activity was nearly abolished by 10 microM siguazodan or 10 microM cyclic GMP (cGMP,) selective inhibitors of the cGMP-inhibited PDE. Kinetic analyses of the enzyme activity contained within this major peak of PDE activity revealed a cAMP Km = 3 microM and a rolipram Ki = 0.5 microM, values characteristic of PDE IV. Additional studies revealed the presence of a small amount of Ca++/calmodulin-stimulated PDE, but no cGMP-stimulated PDE or cGMP-specific PDE activity. In an effort to induce PDE activity in intact U937 cells by producing a sustained increase in cAMP content, cells were treated for 4 hr with salbutamol (1 microM), rolipram (30 microM) or a combination of both agents. The combination of salbutamol and rolipram produced a 2- to 3-fold increase in PDE activity in U937 cells; when used alone, rolipram was without effect whereas salbutamol induced an increase that was approximately one-half of that observed with the combination. Isozyme isolation and characterization revealed that the overall elevation of cellular PDE activity could be accounted for by a 2- to 3-fold increase in the Vmax of PDE IV with no change in its Km. The induction of PDE IV by salbutamol was: 1) concentration- and time-dependent; 2) detectable only after prolonged (2-4 hr) agonist exposure; 3) preceded by an increase in cAMP content and an activation of cAMP-dependent protein kinase; 4) mimicked by 8-bromo-cAMP and prostaglandin E2; 5) reversible within 3 hr of salbutamol removal; and 6) abolished by cycloheximide or actinomycin D. Collectively, these results indicate that the major PDE isozyme in the soluble fraction of U937 cells is PDE IV and that the activity of this enzyme is increased markedly in cells after prolonged exposure to agents that increase cAMP content.
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PMID:Stimulation of beta adrenoceptors in a human monocyte cell line (U937) up-regulates cyclic AMP-specific phosphodiesterase activity. 133 58

Tryptophan hydroxylase (TPH) can be immobilized by adsorption to Pansorbin after binding to the monoclonal antibody PH8. This method yields recoveries of 35%-40% of total TPH activity in crude extracts and can be completed in 1.5 h. The immobilized form of TPH retains the essential kinetic properties of the native enzyme and responds to activators (phosphatidylserine) and inhibitors (catechol compounds) as does the native enzyme. Unlike TPH in brain extracts, immobilized TPH is not activated by calcium-stimulated phosphorylating conditions. When extracts from which TPH has been precipitated, and which contain calcium-calmodulin dependent protein kinase are added to immobilized TPH, the activation of TPH is restored. This method of immobilization of TPH via immune-adsorption allows for the highly specific and rapid preparation of affinity purified TPH that can be used to study the regulation of this enzyme by a variety of effectors, especially protein kinases.
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PMID:Immobilization of tryptophan hydroxylase by immune adsorption: a method to study regulation of catalytic activity. 133 52

1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na(+)-Ca2+ exchange current in inside-out cardiac membrane patches (1-10 G omega seals with 14-24 microns pipette tip diameters) excised from guinea-pig, rabbit and mouse myocytes. 2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads. 3. To further establish the validity of the method for ion transport studies, measurements of Na(+)-K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (Kd, 94 microns), (iii) fast, rigorous concentration control and (iv) Na(+)-K+ pump densities in the range of whole-cell densities. 4. Stimulation of outward Na(+)-Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange current by cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium-independent current. 5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (half-time) of about 3 min. However, the reversal took place much more rapidly (t50, 5-20 s) in patches from individual guinea-pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (Kd, 10-65 microns-free calcium). 6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased. 7. The Kd for MgATP of the initial rate of stimulation of outward exchange current was 3 mM or greater. When decay was rapid, the steady-state dependence of exchange current on MgATP also had a Kd of 3 mM or greater. 8. Stimulation of Na(+)-Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mM-EGTA. 9. The stimulatory effect of 2 mM-MgATP was not inhibited by up to 200 microM of the protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and calcium-calmodulin-dependent protein kinase II.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The giant cardiac membrane patch method: stimulation of outward Na(+)-Ca2+ exchange current by MgATP. 133 2

Rat liver fructose-1,6-bisphosphatase was phosphorylated by cAMP-dependent protein kinase to 2.6 mol phosphate/mol subunit but not by Ca2+/phospholipid-dependent and Ca2+/calmodulin-dependent protein kinases. It was demonstrated that phosphorylation of Ser-341 and Ser-356, and to a much lower extent, Ser-338, was dependent on the presence of intact arginine residues. This observation implicates that the intact three-dimensional structure of the substrate is necessary for phosphorylation of Ser-356 since the closest arginine is located at a six amino acid residue distance.
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PMID:Further studies on the phosphorylation and kinetics of rat liver fructose-1,6-bisphosphatase: importance of the three-dimensional structure of a substrate to protein kinase A. 133 13

Much evidence has accumulated suggesting that neurons in autonomic and dorsal root ganglia possess voltage-dependent currents that link with transmitter receptors through intracellular signal transduction systems. The M current (IM), a voltage-dependent potassium current, was activated at potentials more positive than -65 mV, while the H current (IH), a voltage-dependent nonselective cationic current, was activated at potentials more negative than -50 mV. The hydrolyzable form of ATP was required to activate IM and IH. Intracellular application of calmodulin enhanced the amplitude of IM in a calcium-dependent manner. IM was reduced by W-7, a calmodulin antagonist, and by ML-9, an inhibitor of calmodulin-dependent protein kinase. IH was enhanced by intracellular loading with cyclic adenosine monophosphate (AMP) or bath application of forskolin and membrane-permeable cyclic AMP analogues. Isobutylmethylxanthine also increased the maximal conductance of IH. IH was depressed by H-8 but not by phorbol ester. It is concluded that the resting membrane conductance of these ganglion cells can be regulated by basal activities of calmodulin-dependent protein kinase and A kinase.
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PMID:Cellular metabolism regulating H and M currents in bullfrog sympathetic ganglia. 133 97

The basic cellular mechanisms involved in the regulation of (Na + K)-ATPase are discussed. Various ligands seem to be responsible for the short-term modulation of this enzyme activity (intracellular messengers). Cytosolic Ca2+ has a key role in mediating changes induced by hormones or receptor agonist; but, in turn, intracellular Ca(2+)-dependent proteins like calmodulin, calnaktin or others, are also needed for these changes. Phosphorylation of effector proteins, following the activation of PKC, PKA or CaM-kinase II, may result in changes of (Na + K)-ATPase activity either by a direct effect on the catalytic subunit or by modulating the Na(+)-H+ exchanger thereby resulting in an effect on intracellular sodium, whose concentration is known to be rate-limiting for the enzyme activity. Despite the ubiquity of (Na + K)-ATPase in various organs and tissues, its response to modulators partly depends on the heterogeneity of the alpha-subunit that give rise to the existence of different isoforms. The relative abundance of alpha 1, alpha 2, alpha 3 or other isoforms is tissue-specific and represents another way of regulation among different cell types. While these cellular mechanisms occur in various cell types the kidney shows an opposite response respect to other tissues such as liver or brain. The functional relevance of the mechanisms of acute adaptation of (Na + K)-ATPase, discussed in this review, is becoming increasingly recognized for the renal enzyme, what may contribute to stimulate new approaches to the study of the short-term regulation of the pump activity in molecular terms.
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PMID:Is the renal (Na + K)-ATPase modulated by intracellular messengers? 133 18

Concanavalin A-binding glycoprotein with 250 K M(r) found in the postsynaptic density (PSD)-enriched preparation (or synaptic cytoskeleton) from rat cerebellum was identified with P400 protein from the physicochemical properties and enrichment in the cerebellum. Proteins homologous to the cerebellar 250 K M(r) protein occurred in the PSD-enriched preparations from rat cerebral cortex and from hippocampus, although the contents in the preparations were very low. The 250 K M(r) proteins in the PSD-enriched preparations from cerebellum and from cerebrum were highly phosphorylated by Ca2+/calmodulin (CaM)-dependent protein kinase II. The protein of synaptic plasma membrane (SPM) and PSD-enriched fractions prepared from cerebral cortex were not phosphorylated by the cAMP-dependent protein kinase endogenous to the fractions, whereas the protein from cerebellum was done in SPM and PSD-enriched fractions. The facts suggest that P400 or P400-like protein is closely associated with Ca2+/CaM-dependent protein kinase II in the PSD-enriched preparations, especially in the preparation from cerebral cortex. Phosphorylation of the protein by Ca2+/CaM-dependent protein kinase II may play an important role in the postsynaptic function in both cerebellum and at least in some areas of cerebrum.
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PMID:P400 protein is one of the major substrates for Ca2+/calmodulin-dependent protein kinase II in the postsynaptic density-enriched fraction isolated from rat cerebral cortex, hippocampus and cerebellum. 133 70

Rat parathyroid hormone (PTH) stimulates cAMP-dependent protein kinase and protein kinase C activity in the kidney. However, PTH increases intracellular Calcium in primary cultures of proximal tubular cells. We have investigated the possibility that PTH also stimulates Calcium/calmodulin-dependent protein kinase II (CaM kinase II). We have employed the tandem chromatographic column method, using synthetic peptide as a substrate, to measure the renal CaM kinase II activity. PTH (250 nM) stimulated CaM kinase II activity by about 50% after 15 sec., and activity returned to baseline by 2 min. Calmodulin antagonists significantly impaired the stimulatory action of PTH whereas basal levels of CaM kinase II activity were relatively unaffected. This study demonstrates that PTH does activate CaM kinase II in renal tissue, and suggests another pathway for the actions of PTH in the kidney.
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PMID:Effect of parathyroid hormone on rat renal calcium/calmodulin-dependent protein kinase II. 134 39

Messenger RNA encoding a protein kinase closely related to the catalytic subunit of skeletal muscle phosphorylase kinase has previously been isolated from a human HeLa cell cDNA library, and cross-species Northern hybridization analysis has shown that the rat homolog of this transcript is abundant in the adult testis (Hanks, S.K. (1989) Mol. Endocrinol. 3, 110-116). We now propose that the protein encoded by this transcript be designated as "PhK-gamma T." In this article, the primary structure of the rat homolog of PhK-gamma T is described, as deduced from nucleotide sequences of cDNA and genomic clones. RNase protection analysis reveals that PhK-gamma T transcripts are actually present in a wide variety of adult rat tissues, but at levels 20-100-fold less than what is observed in the testis. In the testis, transcription of the PhK-gamma T gene is initiated at multiple sites as shown by RNase protection and primer extension. Enzymatic activity of PhK-gamma T was demonstrated using renatured bacterially expressed protein. In the presence of Ca2+/calmodulin, PhK-gamma T is able to efficiently phosphorylate glycogen phosphorylase and convert it from an inactive to an active form. We conclude that PhK-gamma T represents a true isoform of phosphorylase kinase catalytic subunit.
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PMID:Molecular cloning and enzymatic analysis of the rat homolog of "PhK-gamma T," an isoform of phosphorylase kinase catalytic subunit. 137 Apr 75


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