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)

1. Stretch-activated anion currents were studied in sino-atrial and atrial cells using the whole-cell patch clamp technique. With continuous application of positive pressure (5-15 cmH2O) through the patch clamp electrode, the cell was inflated and the membrane conductance was increased. 2. Voltage clamp steps revealed that the stretch-activated currents had time-independent characteristics. The increase in membrane conductance was reversible on subsequent application of negative pressure to the electrode. 3. The reversal potential of the stretch-activated currents was shifted by 60 mV for a 10-fold change in intracellular Cl- concentration, while it was unaffected by replacement of Na+ in the extracellular solution by N-methyl-D-glucamine. Cell superfusion with Cl(-)-deficient solution (10 mM Cl-) reduced the amplitude of outward current. These findings indicate that the stretch-activated conductance is Cl- selective. 4. The sequence of anion permeability through the stretch-activated conductance was determined to be I-(1.7) > NO3-(1.5) > Br-(1.2) > Cl-(1.0) > and F-(0.6). SCN- appeared to be more permeant than I-. 5. The stretch-activated conductance was reduced by the Cl- channel blockers, 4,4'-dinitrostilbene-2,2'-disulphonic acid disodium salt, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid or anthracene-9-carboxylate (9-AC). Administration of furosemide or bumetanide had no effect. 6. The stretch-activated Cl- current was recorded even though intracellular Ca2+ ions were chelated by including 10 mM EGTA in the pipette solution. Neither the specific peptide inhibitor of cyclic AMP-dependent protein kinase (50 microM), nor the non-selective blocker of protein kinases, H-7 (20 microM), was effective in reducing the stretch-activated Cl- current, suggesting that the stretch-activated Cl- current is a novel type of cardiac Cl- current, which shows a different modulatory mechanism from that of other cardiac Cl- currents.
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PMID:Stretch-activated anion currents of rabbit cardiac myocytes. 128 78

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.
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PMID:Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts. 138 79

Nitrate acts through the response regulator NarL to activate and repress anaerobic respiratory gene expression in Escherichia coli. The narX gene product encodes a cognate sensor (histidine protein kinase). However, previous work discovered that NarL-mediated nitrate regulation is essentially normal in delta narX deletion mutants. In other two-component regulatory systems studied, the cognate sensor gene is essential for normal regulation. We suggested that NarX-mediated signal transduction reactions are also provided by a functionally redundant nitrate sensor, NarQ. We report here the identification and analysis of narQ insertion mutants. In narX+ strains, a narQ::Tn10 insertion had no perceptible effect on nitrate regulation. However, the same narQ::Tn10 insertion eliminated nitrate regulation when present in delta narX deletion strains. Thus, either narX+ or narQ+ was sufficient for essentially normal NarL-mediated nitrate regulation. The narQ gene mapped to 53 minutes on the E. coli genetic map, a location distinct from all known nitrate regulatory or target genes. The predicted NarQ sequence shares substantial similarity with NarX, particularly in the histidine protein kinase region and in a region of shared similarity with the methyl-accepting chemotaxis proteins. Both NarQ and NarX apparently have N-terminal periplasmic domains, but the primary structures of these regions are largely dissimilar in the two sequences. Analysis of narX* and narL missense alleles in narQ+ versus narQ::Tn10 backgrounds suggests that NarQ and NarX may have subtle functional differences.
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PMID:Either of two functionally redundant sensor proteins, NarX and NarQ, is sufficient for nitrate regulation in Escherichia coli K-12. 152 45

During anaerobic growth, nitrate induces synthesis of the anaerobic respiratory enzymes formate dehydrogenase-N and nitrate reductase. This induction is mediated by a transcription activator, the narL gene product. The narX gene product may be involved in sensing nitrate and phosphorylating NARL. We isolated narX mutants, designated narX*, that caused nitrate-independent expression of the formate dehydrogenase-N and nitrate reductase structural genes. We used lambda narX specialized transducing phage to genetically analyze these lesions in single copy. Two previously isolated narX* mutations, narX32 and narX71, were also constructed by site-specific mutagenesis. We found that each of these alleles caused nitrate-independent synthesis of formate dehydrogenase-N and nitrate reductase, and each was recessive to narX+. The narX* mutations lie in a region of similarity with the methyl-accepting chemotaxis protein Tsr. We suggest that the narX* proteins have lost a transmembrane signalling function such that phosphoprotein phosphatase activity is reduced relative to protein kinase activity.
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PMID:Mutational analysis reveals functional similarity between NARX, a nitrate sensor in Escherichia coli K-12, and the methyl-accepting chemotaxis proteins. 159 21

1. Anion-selective channels from the apical membrane of respiratory epithelia are involved in the secretion of chloride into the airway lumen. In cystic fibrosis (CF) there is an abnormality of phosphorylation-regulated chloride transport in this tissue, whilst a calcium-dependent pathway appears to function normally. 2. Using incorporation of apical membrane vesicles into planar phospholipid bilayers, we have characterized the most commonly seen anion-selective channel from sheep tracheal epithelium. 3. In symmetrical 200 mM-NaCl solutions the channel showed rectification, with a chord conductance at negative voltages of 107 pS and at positive voltages of 67 pS. The channel characteristically demonstrated subconductance states at 1/3 and 3/4 of the fully open level. Selectivity for chloride over sodium was approximately 6:1. 4. The channel required a minimum of approximately 100 microM-calcium on the presumed cytoplasmic surface (cis) for opening events to be observed. Open probability (Po) of the fully open state was markedly voltage dependent, but little effect of voltage was seen on the 1/3 subconductance state. 5. The relative permeabilities of monovalent anions monitored under bi-ionic conditions gave the following sequence: NO3- greater than I- greater than Cl- = Br- much much greater than F-. The order of conductances in symmetrical solutions was Cl- = NO3- greater than Br- greater than I- much much greater than F-. 6. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) produced a dose-related reduction in Po with a flickering block at 10-50 microM and complete block at higher concentrations. 7. ATP produced a dose-related reduction in Po with effects at 1 microM and complete closing at 1 mM. These effects were only seen with addition to the cis chamber. 8. The catalytic subunit of protein kinase A, either when incubated with vesicles prior to incorporation into bilayers, or when added directly to either chamber, produced no effect. 9. Channels with very similar properties were seen from transfected human tracheo-bronchial cells. 10. Recent whole-cell patch-clamp studies have suggested a distinct calcium-activated chloride current in secretory epithelia. The described channel has properties in common with this current and may be a candidate for its single-channel basis.
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PMID:Characterization of a Ca(2+)-dependent anion channel from sheep tracheal epithelium incorporated into planar bilayers. 172 92

Rat liver nuclei protein kinase C is identified as type II isozyme employing monospecific antibodies obtained against each three types of rat brain protein kinase C isozymes. (Yoshida, Y., Huang, F. L., Nakabayashi, H., and Huang, K-P. (1988) J. Biol. Chem. 263, 9868-9873). A major immunoreactive protein band at 80 kDa was revealed by type II isozyme antibodies at each step of purification, nuclear extract included. The nuclear protein kinase C has been purified to apparent homogeneity as revealed by silver nitrate staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing a single 80 kDa protein band. It does seem that 66 kDa protein (Masmoudi, A., Labourdette, G., Mersel, M., Huang, F. L., Huang, K.-P., Vincendon, G., and Malviya, A. N. (1989) J. Biol. Chem. 264, 1172-1179) is a major contaminant devoid of any protein kinase activity. The ratio obtained between protein kinase C enzymatic activity over phorbol dibutyrate bound, at various purification steps, indicates that the nuclear enzyme is a phorbol ester receptor. When isolated nuclei were incubated with 12-O-tetradecanoyl phorbol-13-acetate, endogenous protein kinase C activity was elevated about 8-10-fold suggesting the existence of phorbol ester signaling pathway at the level of nucleus. The role of nuclear protein kinase C is delineated in the regulation of inducible gene transcription
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PMID:Rat liver nuclei protein kinase C is the isozyme type II. 230 97

1. A study has been made of the behaviour of the radiochloride efflux in single muscle fibres from the barnacle, Balanus nubilus. 2. In the majority of the fibres studied, the fractional rate constant for 36Cl efflux is a constant and unaffected by the injection of distilled water (approximately 0 . 3 microliter. in volume). 3. Acidification of the HCO3(-)-containing medium causes stimulation of the Cl efflux, the threshold value being pH 7 . 0. The magnitude of the response is a logarithmic function of the external H+ and HCO3 concentration over a wide concentration range. 4. (i) Total replacement of the external Cl and NO3 fails to alter the course of the Cl efflux. However, the magnitude of the response to acidification is reduced to a marked degree. (ii) Replacement of the external Na by Li reduces not only the Cl efflux but also the size of the response to acidification. 5. Injection of HCl, HCO3 or KCl fails to alter the Cl efflux. Injection, however, of 4 M-KCl or NaCl causes a fall in the efflux. 6. 10( 4)M-ouabain is ineffective. It also fails to alter the response of the Cl efflux to acidification. 7. (i) Injection of cyclic AMP stimulates the Cl efflux in a dose-dependent manner, but only transitorily. (ii) Preinjection of pure protein kinase inhibitor causes a marked reduction in the magnitude of the response to cyclic AMP. 8. Preinjection of pure protein kinase inhibitor fails to affect the response to external acidification. 9. (i) Pretreatment externally with ethacrynic acid reduces the response to external acidification. (ii) External application of 4-acetoamineo-4'-isothiocyano-2,2'-stilbene disulphonate (SITS) reduces the resting Cl efflux. It also abolishes completely the response to acidification. (iii) The effect of 4,4'-diisothiocyano-2,2'-stilbene disulphonate (DIDS) resembles that of SITS. (iv) Injection of H2DIDS fails to reduce the resting efflux but tends to reduce the magnitude of the response to acidification. 10. (i) 5 X 10(-4) M-benzolamide is without effect on the basal Cl efflux. (ii) Benzolamide in high concentration reduces the magnitude of the response to acidification. This occurs within a rather narrow concentration range. 11. (i) A sudden reduction in environmental temperature from 24 to 0 degrees C causes a marked fall in the Cl efflux. (ii) Acidification of the artificial sea water at 0 degrees C stimulates the efflux. 12. The present experiments have led to evidence which is consistent with the view that the Cl efflux is modulated by at least two distinct mechanisms: one is responsive to acidification when HCO3 as buffer is present and involves participation of a benzolamide-sensitive component presumably lying in the fibre membrane. The other is responsive to injection of cyclic AMP, and, and probably involves cyclic AMP-protein kinase.
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PMID:Chloride efflux in single barnacle muscle fibres. 741 34

1. Chloride channels were identified in the basolateral membrane of isolated cortical thick ascending limbs (CTALs) of the mouse nephron by the patch-clamp technique. A channel with a conductance of 45 pS, previously shown to be Cl- selective, was detected in 21% of cell-attached patches when CTAL fragments were pre-incubated with 10 mumol l-4 forskolin for at least 15 min. The same channel was found in only 8.5% of cell-attached patches formed on unstimulated tubules. 2. Another channel with a smaller conductance (7-9 pS) was found in 42.8% of cell-attached patches and 57% of inside-out patches in unstimulated CTAL tubules, but in 82-87% of patches from forskolin-treated tubules. 3. The small channels was Cl- selective (Cl(-)-to-Na+ permeability ratio, PCl/PNa = 9.8) with the permeability sequence: NO3- > Br- > Cl- > F- > gluconate. Channel activity decreased (Br-) or disappeared (NO3-) at negative voltages. At 140 mmol l-1, I- completely inhibited channel activity at all voltages, but a PI/PCl ratio of 1.6 was estimated using a low I- concentration (10 mmol l-1). 4. Internal adenosine triphosphate (ATP) increased normalized current (nPo) in 48% of inside-out patches containing Cl- channels from unstimulated tubules and in 63% of patches from forskolin-treated CTAL tubules. The non-hydrolysable ATP analogue, adenosine 5'-adenylyl imidodiphosphate (AMP-PNP) did not increase channel activity. 5. Adding the catalytic subunit of protein kinase A to the bath in the presence of ATP increased the activity of the small channel in 58% of inside-out patches from unstimulated tubules, but it had no effect on the 45 pS channel. 6. The Cl- channel blockers 5-nitro-2-(3-phenylpropylamine)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) or glibenclamide, all at 0.1 mmol l-1, and diphenylamine-2-carboxylic acid (DPC), at 1 mmol l-1, inhibited the small channel activity by 80-100% in inside-out patches. 7. These results indicate that two Cl- channels with contrasting properties mediate the basolateral step of NaCl absorption in the thick ascending limb of the loop of Henle.
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PMID:A small-conductance Cl- channel in the mouse thick ascending limb that is activated by ATP and protein kinase A. 765 86

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.
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PMID:Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel. 784 Jan 47

Glyceryl trinitrate, isosorbide dinitrate, and isosorbide-5-mononitrate are organic nitrate esters commonly used in the treatment of angina pectoris, myocardial infarction, and congestive heart failure. Organic nitrate esters have a direct relaxant effect on vascular smooth muscles, and the dilation of coronary vessels improves oxygen supply to the myocardium. The dilation of peripheral veins, and in higher doses peripheral arteries, reduces preload and afterload, and thereby lowers myocardial oxygen consumption. Inhibition of platelet aggregation is another effect that is probably of therapeutic value. Effects on the central nervous system and the myocardium have been shown but not scrutinized for therapeutic importance. Both the relaxing effect on vascular smooth muscle and the effect on platelets are considered to be due to a stimulation of soluble guanylate cyclase by nitric oxide derived from the organic nitrate ester molecule through metabolization catalyzed by enzymes such as glutathione S-transferase, cytochrome P-450, and possibly esterases. The cyclic GMP produced by the guanylate cyclase acts via cGMP-dependent protein kinase. Ultimately, through various processes, the protein kinase lowers intracellular calcium; an increased uptake to and a decreased release from intracellular stores seem to be particularly important.
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PMID:Mechanisms of action of nitrates. 787 67


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