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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).
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PMID:ATP counteracts the rundown of gap junctional channels of rat ventricular myocytes by promoting protein phosphorylation. 1008 44

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.
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PMID:Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases. 1045 97

Cytostatin, which is isolated from a microbial cultured broth as a low molecular weight inhibitor of cell adhesion to extracellular matrix (ECM), has anti-metastatic activity against B16 melanoma cells in vivo. In this study, we examined a target of cytostatin inhibiting cell adhesion to ECM. Cytostatin inhibited tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin upon B16 cell adhesion to fibronectin. While the amount of FAK was not affected by cytostatin, electrophoretically slow-migrating paxillin appeared. Alkaline phosphatase treatment diminished cytostatin-induced slow-migrating paxillin. Furthermore, cytostatin increased intracellular serine/threonine-phosphorylated proteins and was found to be a selective inhibitor of protein phosphatase 2A (PP2A). Cytostatin inhibited PP2A with an IC(50) of 0.09 microgram/ml in a non-competitive manner against a substrate, p-nitrophenyl phosphate, but it had no apparent effect on other protein phosphatases including PP1, PP2B and alkaline phosphatase even at 100 microgram/ml. On the contrary, dephosphocytostatin, a cytostatin analogue, without inhibitory effect on PP2A did not affect B16 cell adhesion including FAK and paxillin. These results indicate that cytostatin inhibits cell adhesion through modification of focal contact proteins such as paxillin by inhibiting a PP2A type protein serine/threonine phosphatase. This is the first report that describes a drug with anti-metastatic ability that inhibits PP2A selectively.
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PMID:Cytostatin, an inhibitor of cell adhesion to extracellular matrix, selectively inhibits protein phosphatase 2A. 1055 74

Inside-out patch recordings from rat acutely dissociated cerebral cortical neurons revealed time and voltage-dependent activity of a large-conductance calcium-activated potassium channel. Channel activity inactivated within minutes following a depolarizing voltage step, and was recovered from inactivation by membrane hyperpolarization. Inactivation rate was not influenced by internal calcium or membrane voltage; however, reducing channel activity with intracellular calcium destabilized inactivation. Channel inactivation was abolished by intracellular trypsin treatment, suggesting that an associated inactivating particle was responsible for inactivation. Application of alkaline phosphatase to the internal aspect of the patch membrane increased channel activity and abolished channel inactivation, without affecting its voltage and calcium dependence. Internal application of Mg-ATP, but not Mg-5'-adenylylamidodiphosphate, retarded recovery of channel activity from inactivation, whereas internal application of protein phosphatase-1alpha enhanced recovery from inactivation. The abolition of channel inactivation by alkaline phosphatase was prevented by prior internal tetraethylammonium treatment, indicating that the alkaline phosphatase site is closely associated with the channel pore. These results demonstrate that cortical large-conductance calcium-activated potassium channel inactivation is probably mediated by an endogenous, trypsin-sensitive, inactivation particle. This particle appears to inactivate the open channel and requires a critical phosphate group for stable block. The slow time-course of channel inactivation may have some pathophysiological significance in maintenance of epileptiform activity.
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PMID:Inactivation of large-conductance, calcium-activated potassium channels in rat cortical neurons. 1061 60

Glucose-stimulated and pancreatic islet beta cell-specific expression of the insulin gene is mediated in part by the C1 DNA-element binding complex, termed RIPE3b1. In this report, we define the molecular weight range of the protein(s) that compose this beta cell-enriched activator complex and show that protein phosphatase treatment inhibits RIPE3b1 DNA binding activity. Fractionation of beta cell nuclear extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that RIPE3b1 binding was mediated by a protein(s) within the 37-49-kDa ranges. Direct analysis of the proteins within the RIPE3b1 complex by ultraviolet light cross-linking analysis identified three binding species of approximately 51, 45, and 38 kDa. Incubating beta cell nuclear extracts with either calf alkaline phosphatase or a rat brain phosphatase preparation dramatically reduced RIPE3b1 DNA complex formation. Phosphatase inhibition of RIPE3b1 binding was prevented by sodium pyrophosphate, a general phosphatase inhibitor. We discuss how changes in the phosphorylation status of the RIPE3b1 activator may influence its DNA binding activity.
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PMID:The RIPE3b1 activator of the insulin gene is composed of a protein(s) of approximately 43 kDa, whose DNA binding activity is inhibited by protein phosphatase treatment. 1074 46

We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H(+)-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn(2+), Mn(2+), and Cd(2+)) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 x g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.
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PMID:Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology. 1088 79

Calcineurin was shown previously to be inhibited by members of the tyrphostin family of tyrosine kinase inhibitors, with the most effective inhibition suggested to be caused by the presence of a conjugated side chain (Martin BL, Biochem Pharmacol 56: 483-488, 1998). Retinoids are a family of naturally occurring biomolecules having non-aromatic ring structures and conjugated side chains as substituents on the ring. Three oxidation states of the all-trans configuration of retinoids (retinol, retinal, and retinoic acid) were tested as effectors of calcineurin. Only retinoic acid was found to inhibit calcineurin effectively, with an IC(50) value of approximately 50 microM. Retinol and retinal caused less than 30% inhibition at concentrations up to 100 microM. All three retinoids caused some precipitation of reaction components: retinoic acid and retinal above 50 microM, and retinol above 250 microM. Bacterial alkaline phosphatase was not inhibited by the retinoids, indicating that metal centers alone are insufficient for significant inhibition by retinoic acid. An aromatic ring was not required for inhibition and may not provide additional inhibition, inasmuch as an aromatic analog of retinoic acid (acitretin) showed less effective inhibition. These data are consistent with the presence of conjugated, unsaturated groups enhancing the inhibition of calcineurin.
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PMID:In vitro effect of retinoids on calcineurin activity. 1093 May 34

The pathogenesis of hepatitis C virus (HCV) recurrence after liver transplantation (LT) is poorly understood, but the cellular immune response is likely to have a major role. Daclizumab, an interleukin-2 receptor (IL-2R) antibody that blunts T-cell activation, leading to a decreased risk for cellular rejection, is used frequently in transplant recipients. The aim of this study is to evaluate the effect of daclizumab therapy on the incidence and severity of recurrent HCV. Forty-one liver transplant recipients (21 patients, HCV positive; 20 patients, HCV negative) at high risk for neurological or renal complications of calcineurin inhibitors were administered daclizumab, mycophenolate mofetil (MMF), and steroids in the early post-LT period, followed by tacrolimus and a steroid taper. All patients were followed up prospectively for graft function and disease recurrence with protocol liver biopsies day 7, month 4, and yearly. Compared with patients without HCV, patients with HCV administered daclizumab had greater 4-month serum alkaline phosphatase, total bilirubin, and alanine aminotransferase (ALT) levels. These biochemical differences resolved by 12 months, except for persistent elevation of ALT levels. Compared with a well-matched HCV control population, patients with HCV administered daclizumab were more likely to have an earlier onset of hepatitis, jaundice, and greater histological activity. Recurrent hepatitis progressed more rapidly in the daclizumab group; 45% developed advanced disease within 1 year. HCV viral load in the daclizumab group was significantly greater at both 4 months and 1 year. Results of this study suggest that the use of adjuvant IL-2R antibodies in combination with MMF in the early peritransplantation period may be associated with early recurrence of hepatitis C and more rapid histological progression of disease.
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PMID:Anti-interleukin-2 receptor therapy in combination with mycophenolate mofetil is associated with more severe hepatitis C recurrence after liver transplantation. 1175 8

Bacteriophage lambda protein phosphatase (lambdaPP) with Mn(2+) as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy. Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of lambdaPP, albeit with inhibition constants (K(i)) that range over 5 orders of magnitude. In addition, small organic anions were tested as inhibitors. Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (K(i) = 5.1 +/- 1.6 microM) whereas phosphonoacetic acid (K(i) = 380 +/- 45 microM) and acetohydroxamic acid (K(i) > 75 mM) modestly inhibited lambdaPP. Low-temperature EPR spectra of Mn(2+)-reconstituted lambdaPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme. This suggests a change in the bridging interaction between Mn(2+) ions of the dimer due to protonation or replacement of a bridging ligand. Inhibitor binding also induces several spectral shifts. Hyperfine splitting characteristic of a spin-coupled (Mn(2+))(2) dimer is most prominent upon the addition of orthovanadate (K(i) = 0.70 +/- 0.20 microM) and PhAH, indicating that these inhibitors tightly interact with the (Mn(2+))(2) form of lambdaPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by lambdaPP and other serine/threonine protein phosphatases.
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PMID:Inhibition of bacteriophage lambda protein phosphatase by organic and oxoanion inhibitors. 1179 Jan 29

Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1.Cdc14 complex in vitro by phosphorylation of Net1. We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies.
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PMID:Mass spectrometry-based methods for phosphorylation site mapping of hyperphosphorylated proteins applied to Net1, a regulator of exit from mitosis in yeast. 1209 18


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