Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01189 (beta-endorphin)
 21,003 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Of three casein phosphatases isolated from the cytosol of human cord blood erythrocytes two were cobalt-dependent, E2 and E3. In the presence of CoCl2, E2 activity was the most prominent. In addition to casein, E2 dephosphorylated phosvitin and p-nitrophenyl phosphate (p-NPP) with pH optima at 6.8-7.2 for proteins and 9.0 for p-NPP. The native enzyme had a molecular weight of 104,000 daltons after AcA-44 Ultrogel filtration. According to SDS/PAGE it consisted of two subunits, 78,000 and 15,000 daltons. The 104,000-dalton form exhibited Michaelis-Menten kinetics and had the greatest affinity for casein between protein substrates tested. Ethanol denaturated the enzyme by 80%. Optimal activation of E2 phosphatase was achieved with 5 mmol/l CoCl2 which did not affect the catalytic properties of the enzyme but did affect the rate of 'E-S' complex formation. Inorganic pyrophosphate was not inhibitory for the 104,000-dalton enzyme. Judging by all these properties the natural substrate for E2 casein phosphatase could be P-pyruvate kinase.
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PMID:Cobalt-dependent protein phosphatases from human cord blood erythrocytes. II. Further characterization of E2 casein phosphatase. 283 85

Inhibition of the phosphorylation of the synaptic plasma membrane (SPM) protein B50 by [D-Trp8]-somatostatin in vitro is time-dependent. Increasing the time of incubation of hippocampal synaptic plasma membranes with the peptide from 15 sec to 30 min prior to addition of 7.5 microM [gamma-32P]ATP results in a complete reduction of B50 phosphorylation. Incubation of synaptic plasma membranes for 30 min in the absence of peptide does not alter basal B50 phosphorylation. Neither ACTH nor beta-endorphin produces similar effects--inhibition of B50 phosphorylation by ACTH is maximal at 15 sec and beta-endorphin produces only a small inhibition, even after 30 min. [D-Trp8]-somatostatin is not activating a membrane-bound protease, since maximal inhibition of B50 phosphorylation by the peptide is seen in the presence of leupeptin or bacitracin. Hippocampal synaptic plasma membranes contain protein phosphatase activity. Assays of B50 phosphorylation in synaptic plasma membranes done under conditions that favor either net phosphorylation or dephosphorylation are consistent with inhibition of protein phosphatase activity by [D-Trp8]-somatostatin. This evidence suggests that [D-Trp8]-somatostatin interacts with SPM binding sites in the hippocampus, which may alter the activity of an endogenous protein phosphatase to determine the degree of B50 phosphorylation.
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PMID:Characteristics of [D-Trp8]-somatostatin-sensitive B50 phosphorylation. 287 46

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.
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PMID:Regulation of pigment organelle translocation. II. Participation of a cAMP-dependent protein kinase. 300 26

The effects of immunosuppressant blockers of calcineurin (protein phosphatase 2B) on cAMP formation and hormone release were investigated in mouse pituitary tumor (AtT20) cells. Immunosuppressants enhanced corticotropin-releasing factor- and isoproterenol-evoked cAMP production in proportion with their potency to block calcineurin. Further analysis of cAMP production revealed that intracellular Ca2+ derived through voltage-regulated calcium channels reduces cAMP formation induced by corticotropin releasing-factor or beta 2-adrenergic stimulation and that this effect of Ca2+ is inhibited by blockers of calcineurin. AtT20 cells were found to express at least three species of adenylyl cyclase mRNA-encoding types 1 and 6 as well as a novel isotype, which appeared to be the predominant species. In two cell lines expressing very low or undetectable levels of the novel cyclase mRNA (NCB20 and HEK293 cells respectively), corticotropin-releasing factor-induced cAMP formation was not altered upon blockage of calcineurin activity. These data identify calcineurin as a Ca2+ sensor that mediates the negative feedback effect of intracellular Ca2+ on receptor-stimulated cAMP production. Furthermore, the effect of calcineurin on cAMP synthesis appears to be associated with the expression of a novel adenylyl cyclase isotype, which is highly abundant in AtT20 cells.
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PMID:Calcineurin feedback inhibition of agonist-evoked cAMP formation. 749 91

Recently, the natural vesicant cantharidin was shown to bind exclusively to and inhibit protein phosphatase 2A (PP2A) in mouse tissue extracts (Li and Casida (1992) Proc. Natl. Acad. Sci. USA 89, 11867-11870). To explore the generality of this effect in vesicant action, we measured the protein serine/threonine phosphatase activity in mouse liver cytosol (in the form of the okadaic acid inhibitable increment of p-nitrophenyl phosphate (p-NPP) phosphatase activity) in the presence of aqueous sulfur mustard or its hydrolysis product, bis(hydroxyethyl)sulfide (TDG). Sulfur mustard inhibited p-NPP hydrolysis. However, inhibition correlated with the time elapsed between thawing and the addition of mustard to the enzyme preparation, not with concentration. TDG exhibited a direct, concentration-related inhibition of p-NPP hydrolysis between 30 and 300 microM. We conclude that sulfur mustard also has an inhibitory effect on protein serine/threonine phosphatases. However, the inhibition is an effect of its non-alkykating hydrolysis product TDG, not of sulfur mustard itself.
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PMID:Possible protein phosphatase inhibition by bis(hydroxyethyl)sulfide, a hydrolysis product of mustard gas. 760 98

The properties of the calcium/calmodulin-dependent protein phosphatase calcineurin and its potential role in stimulus-secretion coupling were examined in AtT20 mouse pituitary corticotrope tumor cells. Protein phosphatase activity was assayed by measuring the liberation of 32P from 32P-casein, adrenocorticotropin secretion was measured by radioimmunoassay. About 60% of the total phosphatase activity was inhibited by 500 nM okadaic acid, suggesting the presence of protein phosphatases 1 and/or 2A. A further 25-30% reduction of phosphatase activity was achieved by chelating free calcium. Addition of the EF-hand protein blocker trifluoperazine or a calcineurin autoinhibitory peptide fragment markedly reduced okadaic acid resistant and calcium-dependent protein phosphatase activity indicating that calcium-dependent 32P release is largely due to calcineurin (protein phosphatase 2B). The remaining 10-15% of total activity was Mg2+ dependent and blocked by NaF, hence possibly due to protein phosphatase 2C. Calcineurin activity was inhibited by the immunosuppressants FK506 and cyclosporin A, either when added to the cell lysates or after preincubation of intact cells with the drugs for 30 min at 37 degrees C. When added to lysates, cyclosporin A inhibited calcium/calmodulin-dependent phosphatase more effectively than FK506. However, when tested on intact cells, FK506 proved 10-fold more potent than cyclosporin A. Both immunosuppressive agents enhanced the calcium-dependent release of adrenocorticotropic hormone into the medium, once more, FK506 was 10-fold more potent than cyclosporin A. Taken together, these data suggest that calcineurin is an inhibitory element in the signal transduction pathway controlling exocytotic secretion in pituitary cells that express voltage-operated calcium channels. This is in direct contrast with leukocytes where voltage-operated calcium channels are not found, and calcineurin is an important element for agonist-induced activation.
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PMID:Inhibitory role for calcineurin in stimulus-secretion coupling revealed by FK506 and cyclosporin A in pituitary corticotrope tumor cells. 768 29

Angiotensin II (AII) receptors are known to interact with two distinct guanine nucleotide binding proteins, Gq/11 and Gi, in rat adrenal glomerulosa cells to activate phospholipase C and to inhibit adenylate cyclase, respectively. However, in cultured bovine glomerulosa cells AII potentiates rather than inhibits the stimulatory effect of adrenocorticotropin (ACTH) on cAMP levels. This effect of AII was partially mimicked by phorbol 12-myristate 13-acetate (PMA) and was partially inhibited by staurosporine or depletion of protein kinase C but was unaffected by pertussis toxin treatment. No potentiation was detectable in disrupted cells or in membrane preparations. In intact glomerulosa cells, treatment with cyclosporin A or FK506 completely inhibited AII- or PMA-induced potentiation of cAMP production without affecting the response to ACTH. In COS-7 cells transfected with the rat AT1 receptor, AII caused 2-3-fold enhancement of the ACTH-induced cAMP response, an effect that was partially reproduced by PMA. These potentiating actions of AII and PMA were prevented by preincubation with cyclosporin A or FK506, and the latter effect was abolished by rapamycin. These results implicate the Ca2+- and calmodulin-dependent protein phosphatase, calcineurin, in AII-induced enhancement of adenylate cyclase activity in both adrenal glomerulosa and transfected COS-7 cells. The finding that AII enhances ACTH-stimulated production of cAMP by a second messenger-mediated mechanism that involves the participation of calcineurin reveals an additional mode of cross-talk between pathways activated by Ca(2+)-mobilizing and cAMP-generating receptors.
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PMID:Evidence for participation of calcineurin in potentiation of agonist-stimulated cyclic AMP formation by the calcium-mobilizing hormone, angiotensin II. 792 24

Although cocaine shares the ability of fenfluramine to inhibit the synaptic reuptake of serotonin, previous observations from our group suggest that the genomic effects of fenfluramine in the rat striatum are primarily mediated by dopaminergic rather than serotonergic mechanisms. To compare and further understand the nerve cell type(s) targeted by psychotropic drugs, we studied, by use of immunocytochemistry and in situ hybridization, changes in c-fos in brain nerve cells of the caudate putamen and hypothalamus following acute cocaine or fenfluramine exposure. Predictably, both drugs (20 mg/kg; i.p.) evoked rapid but transient increases in c-fos in the caudate putamen. In addition, double labeling immunocytochemistry indicated that Fos-like protein was expressed preferentially in striatal neurons containing the protein phosphatase inhibitor, DARPP-32. In contrast, fenfluramine, but not cocaine, elicited c-fos mRNA and Fos-like protein in the neuroendocrine paraventricular nucleus (PVN) of the hypothalamus despite the fact that both drugs are known to be equally capable to stimulate the hypothalamic-pituitary-adrenal (HPA) axis. This difference is discussed in terms of serotonergic, dopaminergic and DARPP-32 input to hypothalamic neurons and tanycytes associated with adrenocorticotropin hormone (ACTH) secretion. To further identify the phenotypes of nerve cells expressing c-fos by fenfluramine in the PVN, it was demonstrated that the immediate-early gene was induced in a subpopulation of neurons constitutively expressing nitric oxide synthase (NOS). Taken together, we identified a number of common and disparate actions of cocaine and fenfluramine in striatal and hypothalamic tissue, thereby suggesting that c-fos induction in these two brain structures is differentially regulated by intrinsic events in addition to neuronal phenotype. We propose that the genomic effects produced by these two drugs represent part of a general dopaminergic and glutamateric mechanism by which monoamine reuptake inhibitor drugs affect specific brain nerve cells.
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PMID:Induction of c-fos in rat brain by acute cocaine and fenfluramine exposure: a comparison study. 806 90

Cyclosporin A (CSA), a potent immunosuppressive drug, has recently been shown to bind with high affinity to the immunophilin, cyclophilin. Calcineurin, the calcium-dependent protein phosphatase, binds the cyclophilin/CSA complex, rendering it inactive and blocking dephosphorylation of phosphoproteins. Very high concentrations of cyclophilin have been reported in the brain with a localization identical to that of calcineurin. We have reported that interleukin-2 (IL-2) releases corticotropin-releasing hormone (CRH) by generation of nitric oxide (NO). Nitric oxide synthase (NOS), the enzyme in nitric oxidergic neurons that converts arginine into citrulline plus NO, is inactive in the phosphorylated state. We hypothesized that cyclosporin might therefore inhibit IL-2-induced acute CRH release by blocking the dephosphorylation of NOS by calcineurin. Consequently, we examined the effect of CSA on the release of CRH from mediobasal hypothalami (MBH) in vitro in 'basal' conditions and in the presence of IL-2, which we had previously shown to stimulate CRH release acutely in this preparation. Incubation of MBH for 30 min with IL-2 (10(-13) M), the concentration that was most effective in previous experiments, evoked a significant release of CRH. CSA at 10(-6) or 10(-8) M did not alter basal release of CRH; however, addition of either concentration completely blocked the IL-2-induced release of CRH. This acute action of CSA within the brain is probably mediated by blockade of the dephosphorylation of NOS by calcineurin.
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PMID:Cyclosporin A inhibits interleukin-2-induced release of corticotropin-releasing hormone. 852 89

The protein phosphatase inhibition assay for okadaic acid, the major DSP toxin, modified to use the fluorescence substrates methylumbelliferyl phosphate (MUP) and fluorescein diphosphate (FDP), was compared to the assay using p-nitrophenylphosphate (p-NPP) and the bioluminescence assay using luciferin phosphate (L-P). Under the standard assay conditions used okadaic acid inhibited the enzyme activity dose-dependently with IC50 values of 1.5 nM (MUP) and 1.2 nM (FDP). This compares to IC50 values of 0.9 and 6 nM using L-P and p-NPP respectively. CDP-star, a chemiluminescence substrate, was not hydrolysed by the enzyme. Decreasing the enzyme concentration lowered the IC50 for the colorimetric method (IC50=2 nM [p-NPP], 0.75 nM enzyme) but no shift was observed with fluorimetry. However at enzyme concentrations < 1.5 nM (standard assay) the error margin was too great for routine analysis. The method using fluorimetry allowed detection of okadaic acid concentrations to levels < or = 1 microg/100 g of mussel tissue which is well below the limit of 20 microg/100 g (mouse bioassay) set by some regulatory agencies. Determination of the toxin content in naturally contaminated mussels in three separate experiments gave coefficients of variance ranging from 16 to 29% (MUP) and from 8 to78% (p-NPP). Multicomparison studies showed that concentrations of okadaic acid in naturally contaminated mussel samples determined by fluorescence generally agreed with those obtained using ELISA and LC-MS procedures, and with the mouse bioassay. However using the mouse bioassay as the standard, values determined by the ELISA, PP-2A and LC-MS all scored false negative results compared to those for the mouse bioassay in the range 20-40 microg/100 g mussel, and at the limit of the mouse bioassay the values by the other three methods were substantially less. With few exceptions the methods scored okadaic acid with highest to lowest values in the following order: mouse bioassay > ELISA > PP-2A > LC-MS. The fluorimetric assay was both more sensitive and accurate than the colorimetric assay (the latter showed a propensity towards false positives in the region 20 microg/100 g), and the moderate increase in equipment cost appears to be outweighed by the performance of the method.
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PMID:Evaluation of the fluorometric protein phosphatase inhibition assay in the determination of okadaic acid in mussels. 1034 Aug 30


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