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)

The ability of phosvitin and histone to serve as substrates for possible protein kinase(s) at the cell surface was examined with regard to their suitability as indicators of such activity. While phosvitin did not interfere with the membrane barrier of HeLa cells, 3T3 and SV 3T3 cells, histone caused severe damage as indicated by both uptake of viability stains Trypan Blue and diamidino-phenylindol and by release of intracellular compounds such as lactate dehydrogenase, cAMP-dependent protein kinase(s), and metabolically prelabelled proteins. Where intactness of the cell membrane is prerequisite for verification of ecto-protein kinase, histone cannot be used as substrate. In contrast, we found that phosvitin is suitable for assays of cell surface located protein kinase activity.
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PMID:Assays of cell surface protein kinase: importance of selecting cytophilic substrates. 706 8

A chloromethyl ketone derivative of lactic acid is a potent inhibitor of glycolysis of Ehrlich ascites tumor cells. It inhibited glycolysis of intact cells by about 50% at 200 microM (100 nmol/mg of protein) while cell-free extracts were inhibited 50% at 50 microM (50 nmol/mg of protein). N alpha-(p-Tosyl)-L-lysine chloromethyl ketone and N alpha-(p-tosyl)-L-phenylalanine chloromethyl ketone inhibited only slightly or not at all at this concentration. The inhibition was localized at the hexokinase and phosphofructokinase steps since these two enzymes added to an inactivated extract restored the glycolytic activity, whereas none of the other glycolytic enzymes did. In fact, addition of pyruvate kinase or lactate dehydrogenase, which stimulated glycolysis, resulted in a more pronounced inhibition. Glycolysis and hexokinase activities in extracts of Rous sarcoma virus transformed cells were considerably more sensitive to the inhibitor than the activities from normal chick embryo fibroblasts. Hexokinase from mouse brain required 50 times higher concentrations for inhibition than the enzyme from mouse Ehrlich ascites tumor cells. Yeast hexokinase was unaffected at all concentrations tested. Since 5,5'-dithiobis(2-nitrobenzoate) protected against the inhibition, the chloromethyl ketone appeared to inhibit by interaction with an essential SH group. A pronounced inhibition of protein kinase activity of plasma membranes of Ehrlich ascites tumor cells was observed in the presence of the chloromethyl ketone. As in the case of glycolysis, the chloromethyl ketone of lactic acid was a more potent inhibitor of protein kinase activity than several other chloromethyl ketones that were tested.
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PMID:Inhibition of hexokinase and protein kinase activities of tumor cells by a chloromethyl ketone derivative of lactic acid. 710 7

Rabbit gastric glands were treated with alpha-toxin to test for permeabilization of basolateral membrane and retention of functional activity of parietal cells. Treatment with up to 400 U alpha-toxin/mL resulted in a dose-dependent increase in permeabilization, as judged by nuclear uptake of trypan blue (960 daltons), while causing relatively little loss of cytoplasmic macromolecules in the size range of lactate dehydrogenase (134,000 daltons). In the presence of cAMP and ATP, alpha-toxin-permeabilized resting gastric glands were stimulated to accumulate aminopyrine by approximately 10-fold over glands incubated without added nucleotides. Aminopyrine accumulation in stimulated permeabilized glands was inhibited by specific H+,K(+)-ATPase inhibitors, omeprazole and SCH-28080, and by the selective inhibitor of protein kinase A, H-89 (IC50 = 7.17 +/- 2.05 microM; n = 4). Aminopyrine accumulation in the alpha-toxin-treated glands was dependent on both exogenous ATP and cAMP; however, when no exogenous ATP was present, cAMP-activated aminopyrine accumulation reached approximately 50% of maximum, and at levels of ATP > 0.05 mM, maximal aminopyrine accumulation occurred without exogenous cAMP. In the presence of ATP alone, aminopyrine accumulation in permeabilized glands achieved 61.1 +/- 3.2% (n = 10; range, 50-70%) of the values measured on paired samples of intact glands stimulated with histamine plus isobutylmethylxanthine. These results demonstrate the functional responsiveness of alpha-toxin-permeabilized resting gastric glands. The participation of a protein kinase A dependent pathway during activation of permeabilized parietal cell is proposed.
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PMID:Acid secretion in alpha-toxin-permeabilized gastric glands. 752 Jul 7

Treatment of ovine pars tuberalis (oPT) cultures with forskolin activates adenylyl cyclase, leading to increased levels of cyclic AMP, activation of protein kinase A, phosphorylation of the calcium/cyclic AMP response-element binding protein and the increased synthesis and secretion of several proteins. Simultaneous treatment with melatonin inhibits or reverses these effects of forskolin. In the neonatal rat pituitary, the inhibitory effects of melatonin are mediated by changes in membrane potential. This study therefore investigated whether the inhibitory action of melatonin in oPT cultures is also dependent on the modulation of plasma membrane potential. Treatment of cultures with the ionophore valinomycin selectively permeabilised the cell plasma membrane to potassium, thereby causing membrane hyperpolarisation. In cultures of oPT, valinomycin inhibited in a concentration-dependent manner (maximal effect 2 microM) the stimulatory action of forskolin (1 microM) on intracellular levels of cyclic AMP, indicating that the activity of adenylyl cyclase in this tissue is sensitive to hyperpolarisation of the plasma membrane. However, increasing the extracellular concentration of potassium from 5 mM to 100 mM, which would depolarise the plasma membrane, had no effect on the inhibitory action of melatonin (1 microM) in forskolin-stimulated cultures. This indicated that melatonin could be effective in cells with sustained depolarisation. To test directly whether integrity of the plasma membrane is essential for melatonin to inhibit adenylyl cyclase, cultures were treated with the cholesterol-chelating agent saponin (50 micrograms/ml). Saponin increased cellular permeability to trypan blue and enhanced the release of the cytoplasmic enzyme lactate dehydrogenase to the extracellular medium, demonstrating that cell plasma membranes had been permeabilised, thereby abolishing membrane polarity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The inhibitory action of melatonin in the ovine pars tuberalis is not dependent on changes in plasma membrane potential. 763 31

The effects of lysophosphatidylcholine (LPC), a vasoactive phospholipid, on intracellular free calcium concentration ([Ca2+]i), DNA synthesis and cytotoxicity of vascular smooth muscle cells (VSMC) were studied. LPC from 10(-7) to 10(-5) mol/l dose-dependently induced a sustained increase in [Ca2+]i. In contrast to the response of [Ca2+]i induced by angiotensin II, that induced by LPC was totally abolished when extracellular Ca2+ was removed, was not affected by pretreatment of the cells with islet-activating protein, and was not desensitized by repeated addition. 8-(N,N-Diethylamino)octyl 3,4,5-trimethoxybenzoic acid (TMB-8), an inhibitor of Ca2+ release from intracellular Ca2+ stores, 1-(5-isoquinolinesulfonyl)-2-methylpiperadine dihydrochloride (H-7), an inhibitor of protein kinase C, KT5823, an inhibitor of protein kinase G, and Ca2+ channel blockers failed to suppress the LPC-induced increase in [Ca2+]i. LPC at 10(-5) mol/l caused significant stimulation of [3H]thymidine incorporation into VSMC, and at concentrations of 10(-5) mol/l and higher dose-dependently stimulated release of lactate dehydrogenase in cell culture supernatants. Moreover, digitonin mimicked the effects of LPC on [Ca2+]i, and also caused similar effects to those of LPC on DNA synthesis and cytotoxicity in VSMC. These observations suggest that LPC causes both cell growth and cell injury of VSMC, at least partly, through its detergent action, causing membrane leakiness and resultant [Ca2+]i overload in vitro, thus indicating the possible participation of LPC in atherosclerosis and/or injury of the vascular wall.
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PMID:Lysophosphatidylcholine causes Ca2+ influx, enhanced DNA synthesis and cytotoxicity in cultured vascular smooth muscle cells. 777 68

O2 plays a dominant role in the metabolism and viability of cells; changes in O2 supply lead to many physiological responses in the cell. Recent reports have shown that hypoxia induces the transcription of a number of genes, among them those for the glycolytic enzymes. We have investigated signalling events that may lead to enhanced activity of lactate dehydrogenase (LDH) in cultured vascular smooth muscle (VSM) cells derived from rat aorta, grown under hypoxic conditions (1% versus 20% O2). LDH was chosen because this enzyme exhibits one of the largest increases in activity among the glycolytic enzymes after hypoxic stimulation of cells. Hypoxic exposure of VSM cells for 24 h resulted in a 2-fold increase in LDH activity and in a 2.5-fold increase in intracellular cAMP levels. Agents that activate adenylate cyclase, such as forskolin, cholera toxin and 1-methyl-3-isobutylxanthine (IBMX), and thus increase cAMP production, significantly induced LDH activity. Moreover, induction of LDH activity by hypoxia was prevented in the presence of the protein kinase A inhibitor N-[2-(methyl-amino)ethyl]-5-isoquinolinsulphonamide dihydrochloride (H-8), and the cyclooxygenase inhibitor indomethacin. In contrast to the cAMP-stimulating agents, stable cGMP analogues (dibutyryl-cGMP, 8-bromo-cGMP), activators of protein kinase C [12-O-tetradecanoylphorbol-13-acetate (TPA), and 1-oleoyl-2-acetyl-glycerol (OAG), and the calcium ionophore ionomycin did not alter LDH activity in VSM cells kept at 20% O2. A dose-dependent increase in LDH activity was also observed in normoxic cells exposed to cobalt chloride (50-200 microM), indicating that a metal binding protein might be involved in this signalling cascade.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hypoxia and cobalt stimulate lactate dehydrogenase (LDH) activity in vascular smooth muscle cells. 789 7

The vasoactive peptide endothelin-1 (ET-1) dose-dependently increased release of 51Cr from human cerebromicrovascular endothelial cells (HBEC), without affecting cell viability as assessed by lactate dehydrogenase release. ET-1 also induced transient accumulation of inositol triphosphate (IP3) and release of [3H] arachidonic acid (AA) from HBEC. The ET-1-induced 51Cr release, formation of IP3, and AA release from HBEC were competitively inhibited by selective ETA subtype receptor antagonist BQ-123. ET-1-stimulated 51Cr- and AA release from HBEC were potentiated by proteinkinase C (PKC) activator phorbol-myristate ester, and abolished by H7, an inhibitor of PKC. Dexamethasone, indomethacin, acetylsalicylic acid, imidazole, as well as the inhibitor of protein kinase A, H8, had no effect on 51Cr release. The results suggest that ETA-receptor mediated activation of PKC and increase in the HBEC 'permeability' for low molecular weight molecules in response to excessive release of endothelins from either HBEC or surrounding tissues during pathologic conditions may contribute to the formation of cerebral edema.
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PMID:Arachidonic acid release and permeability changes induced by endothelins in human cerebromicrovascular endothelium. 797 60

The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Sp1 binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consensus cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of the transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5'-flanking region. Transient transfection analyses of a -1173/+25 bp LDH A-chLoramphenicol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcription. Deletion of the -1173/-830 bp sequence restored basal and cyclic AMP (cAMP)-inducible activity. Point mutations in the Sp1 binding sites of a -830/+25 bp promoter fragment reduced basal but not the relative degree of cAMP-inducible activity. cAMP-regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -48/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65%. The CRE binds a 47 kDa protein which has previously been identified as CRE binding protein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that expresses the catalytic subunit of cAMP-dependent protein kinase stimulates LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB-327. This study emphasizes a fundamental role of several modules including Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.
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PMID:Analysis of the rat lactate dehydrogenase A subunit gene promoter/regulatory region. 799 73

Changes between serial laboratory test results can be significant, even if none of the individual results exceeds the reference interval. We developed a statistical method for the calculation of reference change limits from routine patients' data for situations in which the majority of the patients can be considered suitable reference subjects. The method was applied to cardiac enzyme data [creatine kinase (CK; EC 2.7.3.2), creatine kinase isoenzyme MB (CK-2), lactate dehydrogenase (LD; EC 1.1.1.28), and lactate dehydrogenase isoenzyme 1 (LD-1)] from 2029 consecutive patients. We used hospital discharge diagnoses to exclude patients with the diagnosis of myocardial infarction or myocarditis, but we also studied the characteristics of the method on unselected patients' data. The reference change limits derived from the diagnosis-selected patient group were as follows (U/L, activity measurements in serum at 37 degrees C according to Scandinavian recommendations): CK from -39 to 27, CK-2 from -8 to 7, LD from -86 to 85, and LD-1 from -19 to 15. Similar limits were obtained by conventional statistical methods from a group of 29 hospitalized patients with no myocardial symptoms. Our results suggest that it is possible to produce clinically applicable reference change limits from routine data.
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PMID:Method for determining reference changes from patients' serial data: example of cardiac enzymes. 822 24

The polymorphonuclear granulocyte, or neutrophil, has been implicated as a mediator of tissue-destructive events because it releases the preformed proteolytic enzymes elastase and cathepsin G, and, as a result of myeloperoxidase action, hypochlorous acid. We show that elastase inactivates and fragments creatine kinase isoenzymes CK-2 and CK-3, and, to a lesser extent, lactate dehydrogenase (LD) isoenzyme LD-1, whereas cathepsin G acts only on CK-2. Both neutrophil enzymes act on LD-3. The course of inactivation was followed by measuring the loss of catalytic activity at 37 degrees C. The evidence for fragmentation was obtained by gel filtration; electrophoresis after sample treatment with sodium dodecyl sulfate and 2-mercaptoethanol was less satisfactory for this purpose. Hypochlorous acid inactivates CK activity by about 75% at concentrations as low as 8 mumol/L and totally at concentrations > 140 mumol/L, whereas LD activity is not affected until concentrations exceed 200 mumol/L. After a myocardial infarction, the number of neutrophils increases; they are triggered and concentrate around damaged myocardial tissue. Our data suggest that neutrophils may inactivate and fragment "cardiac" enzymes released from such damaged tissue.
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PMID:In vitro effect of elastase and cathepsin G from human neutrophils on creatine kinase and lactate dehydrogenase isoenzymes. 828 31


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