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)

An in vitro model to study the molecular control of binding of highly purified synaptic vesicles to presynaptic plasma membranes has been developed. Presynaptic plasma membranes were immobilized by dotting onto nitrocellulose, and binding of iodinated synaptic vesicle membranes was studied under varying experimental conditions. Synaptic vesicles bind to presynaptic plasma membranes in the presence of Ca2+ and ATP. Binding is reduced in the presence of EGTA and abolished by the calmodulin antagonist trifluoperazine. Vesicle binding is stimulated 5-fold after incubation--prior to dotting--of presynaptic plasma membranes with ATP in the presence of the phorbol-ester 12-O-tetradecanoylphorbol-13-acetate (1 microM) and 2.5-fold after preincubation with Ca2+ (50 microM). Pretreatment of plasma membranes with alkaline phosphatase strongly reduces vesicle binding. Microsomes prepared from bovine liver did not bind to presynaptic plasma membranes. Our results suggest that activation of protein kinase C and Ca2+ stimulate binding of synaptic vesicles to the presynaptic membrane. In the intact nerve terminal this interaction may represent an initial step in synaptic vesicle exocytosis.
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PMID:In vitro binding of isolated synaptic vesicles to presynaptic plasma membranes: activation by Ca2+ and protein kinase C. 130 19

We have demonstrated that the alpha 2,3 sialyltransferase (alpha 2,3 ST) from C6 cultured glioma cells was inhibited in vivo by W-7 and related Ca2+/Calmodulin (Ca/CaM) antagonists while protein kinase C effectors had no effect. Dephosphorylation of alpha 2,3 ST by the wide specificity alkaline phosphatase led to inactivation indicating that the enzyme is phosphorylated. The serine/threonine protein phosphatase inhibitors okadaic acid and Calyculin A led also to an inhibition of alpha 2,3 ST activity. In addition, Ca/CaM antagonists and phosphatase inhibitors led both to an inhibition of a alpha 2,3 sialoglycoprotein from C6 glioma cells as demonstrated with lectin affinity blotting. A concerted regulatory mechanism with phosphorylation/dephosphorylation of alpha 2,3 ST is then postulated.
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PMID:Study of O-glycan sialylation in C6 cultured glioma cells: regulation of a beta-galactoside alpha 2,3 sialyltransferase activity by Ca2+/calmodulin antagonists and phosphatase inhibitors. 132 69

1. Two distinct patterns of Ca(2+)-mediated activation of Ca(2+)-ATPase were identified in calmodulin-depleted membranes. 2. In membranes showing no activation (type A), preincubation with micromolar concentration of cyclic AMP and ATP made possible stimulation of the enzyme while in membranes already exhibiting activation (type B), preincubation with cyclic AMP and ATP abolished the activation. 3. ATPase stimulation in type A membranes was suppressible by leupeptin. 4. Triton extractable inhibitor isolated from type A membranes was as active as that derived from type B membranes only after preincubating the membranes with cyclic AMP and ATP. 5. The inhibitor could be inactivated by alkaline phosphatase.
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PMID:Variations in Ca(2+)-mediated activation of Ca(2+)-ATPase and its associated inhibitor in erythrocyte membrane. 132 91

We performed phosphate analysis of tau proteins isolated from normal human brain, tau proteins associated with paired helical filaments (PHF-tau), and Alzheimer tau not associated with PHF. These tau fractions were of high purity. Normal and Alzheimer tau were purified by heat treatment, acid extraction and calmodulin-affinity chromatography with or without HPLC. Fractions containing primarily PHF-tau polypeptides of 60, 64 and 68 kDa and their degraded fragments were purified either on a sucrose density gradient as filaments (PHF) or by heat treatment and acid extraction as amorphous proteins (PHF-tau). PHF and PHF-tau were found to contain 6-8 mol phosphate/mol protein while normal and Alzheimer tau proteins contained 1.9 and 2.6 mol phosphate/mol protein, respectively. Upon 2-h incubation with alkaline phosphatase, PHF lost two of the phosphate groups without apparent changes in the stability and morphology of PHF. The released phosphate originated from the N-terminal half of PHF-tau as determined by immunoblotting with antibodies to epitopes blocked by phosphorylation. Tau-1 and E-2, and by a prominent shift in the electrophoretic mobility of some fragments of PHF-tau. The shift in mobility was not observed with the C-terminal fragments of 25-26 kDa, which retained the epitope to Tau 46. The results suggest that the phosphorylation sites not affected by phosphatase may be located in the 25-26 kDa C-terminal region of PHF-tau and may play a role in structural stability of PHF.
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PMID:Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments. 147 94

The rate of inactivation of the voltage-dependent Ba2+ current in dissociated neurons from the snail Helix aspersa was found to be modulated by phosphorylation. Conditions were chosen such that the most likely mechanism of inactivation of the Ba2+ current was a voltage-dependent/calcium-independent inactivation process. If adenosine-triphosphate (ATP) was not included in the patch electrode filling solution, or if alkaline phosphatase was added, the Ba2+ current rapidly ran down and the rate of inactivation greatly increased with time. Dialysis with either ATP gamma S or the phosphatase inhibitor okadaic acid (OA) either enhanced the amplitude or greatly reduced the rate of run-down of the Ba2+ current (depending upon the presence of ATP), as well as reducing the rate of inactivation. However, dialysis with either the catalytic subunit of the cyclic-adenosine-mono-phosphate-dependent protein kinase (cAMP-PK), a synthetic peptide inhibitor of this enzyme, or staurosporine (a potent inhibitor of protein kinase C), did not have any significant effect on the amplitude or kinetics of the Ba2+ current. Surprisingly, dialysis with a peptide inhibitor (CKIP) of the Ca2+/calmodulin-dependent protein kinase II (Ca(2+)-CaM-PK) significantly reduced the rate of inactivation of this current. These results suggest that phosphorylation may exert its effect by modulating the gating properties of the Ca2+ channels.
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PMID:Inactivation of the Ba2+ current in dissociated Helix neurons: voltage dependence and the role of phosphorylation. 161 19

mAbs specific for calmodulin were used to examine the distribution of calmodulin in vegetative Dictyostelium cells. Indirect immunofluorescence indicated that calmodulin was greatly enriched at the periphery of phase lucent vacuoles. The presence of these vacuoles in newly germinated (non-feeding) as well as growing cells, and the response of the vacuoles to changes in the osmotic environment, identified them as contractile vacuoles, osmoregulatory organelles. No evidence was found for an association of calmodulin with endosomes or lysosomes, nor was calmodulin enriched along cytoskeletal filaments. When membranes from Dictyostelium cells were fractionated on equilibrium sucrose density gradients, calmodulin cofractionated with alkaline phosphatase, a cytochemical marker for contractile vacuole membranes, at a density of 1.156 g/ml. Several high molecular weight calmodulin-binding proteins were enriched in the same region of the gradient. One of the calmodulin-binding polypeptides (molecular mass approximately 150 kD) cross-reacted with an antiserum specific for Acanthamoeba myosin IC. By indirect immunofluorescence, this protein was also enriched on contractile vacuole membranes. These results suggest that a calmodulin-binding unconventional myosin is associated with contractile vacuoles in Dictyostelium; similar proteins in yeast and mammalian cells have been implicated in vesicle movement.
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PMID:Association of calmodulin and an unconventional myosin with the contractile vacuole complex of Dictyostelium discoideum. 162 38

Soybean nodulin-26, a homologue of bovine eye lens major intrinsic protein (MIP-26), is an integral protein of the peribacteroid membrane in symbiotic root nodules. It comprises 271 amino acids with six potential transmembrane domains and lacks an amino-terminal signal sequence. A full-length nodulin-26 cDNA and its various deletion derivatives were transcribed in vitro after linking them to bacteriophage T3 promoter. In vitro translation of these transcripts in a rabbit reticulocyte lysate, in the presence or absence of canine pancreatic microsomal membranes, suggested that nodulin-26 is cotranslationally inserted into the microsomes without a cleavable signal peptide. The first two transmembrane domains (103 amino acids) of the protein are sufficient for microsomal membrane insertion. Membrane-translocated nodulin-26 binds to Con-A and is sensitive to endoglycosidase-H treatment, suggesting that it is glycosylated. Native nodulin-26 from root nodules retains its sugar moiety as it, too, binds to Con-A. Chemical cleavage mapping at cysteine residues, a trypsin protection assay, and the Con-A binding affinity of nodulin-26 suggested that both the NH2 and COOH termini of this protein are on the cytoplasmic surface of the peribacteroid membrane, while the glycosidic residue is on the surface of the membrane facing the bacteroids. In vitro phosphorylation experiments showed that nodulin-26 is a major phosphorylated protein in the peribacteroid membrane. This phosphorylation is mediated by a Ca(2+)-dependent, calmodulin-independent protein kinase located in the peribacteriod membrane. Externally supplied acid phosphatase dephosphorylates this protein, but alkaline phosphatase does not. Based on its homology with several eukaryotic and prokaryotic channel-type membrane proteins, nodulin-26 may form a channel translocating specific molecules to the bacteroids during endosymbiosis in legume plants.
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PMID:Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane. 162 42

We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full-term human placenta. These purified membranes were enriched 25-fold in Na+/K(+)-adenosine triphosphate (ATPase), 37-fold in [3H] dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis-Menten kinetics, with a Km Ca2+ of 0.18 +/- 0.05 microM and Vmax of 0.93 +/- 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration-dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver-Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP-independent Ca2+ uptake was not sensitive to 10(-6) M nitrendipine nor to 10(-4) M verapamil. An ATP-dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 +/- 0.02 microM and Vmax 3.4 +/- 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10(-6) M calmodulin strongly enhanced the initial ATP-dependent Ca2+ uptake. Finally, no Na+/Ca2+ exchange process could be demonstrated.
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PMID:Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast. 165 Mar 72

In preparations of synaptic terminals (synaptosomes) isolated from rat brain, the activity of phospholipase A2 (PLA2), a phospholipid hydrolase that serves a central function in signal transduction, was inhibited in a Ca(2+)-dependent manner by incubation with 60 mM K+ or with the Ca(2+)-selective ionophore ionomycin. Reversal by alkaline phosphatase treatment suggested that this inhibitory effect resulted from phosphorylation of a synaptosomal protein substrate. When lysed synaptosomes were incubated with Ca2+/calmodulin (CaM), purified Ca2+/CAM-dependent protein kinase II (Ca2+/CaM-dependent PK II) and ATP, PLA2 activity in lysates was nearly abolished within 10 min. This effect was accompanied by a marked decrease in the Vmax of the enzyme and little or no change in the Km. Furthermore, Ca2+/CaM with ATP but without exogenous Ca2+/CaM-dependent PK II partially inhibited PLA2 activity, and this effect was prevented by treating the lysates with a selective peptide inhibitor of Ca2+/CaM-dependent PK II. In contrast, incubation of intact synaptosomes with 4 beta-phorbol 12-myristate 13-acetate or of lysed synaptosomes with purified protein kinase C had little or no effect on PLA2 activity. The results strongly suggest that the Ca(2+)-dependent inhibition of PLA2 activity observed in intact nerve endings was produced by activation of the multifunctional Ca2+/CaM-dependent PK II. A membrane-permeable adenylyl cyclase activator, forskolin, enhanced PLA2 activity in intact synaptosomes, and cAMP-dependent protein kinase potentiated PLA2 activity in lysed synaptosomes. Furthermore, another broad-spectrum protein kinase present in synaptic terminals, casein kinase II, also potentiated PLA2 activity in lysed synaptosomes. The effects of both protein kinases were associated with a decrease in Km and no change in Vmax. The results suggest that PLA2 activity in synaptic terminals is subject to bidirectional control by distinct signal transduction pathways. Moreover, mutually antagonistic effects of the Ca2+/CaM-dependent PK II and PLA2 pathways provide a possible molecular mechanism for bidirectional modulation of neurotransmitter release.
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PMID:Bidirectional control of phospholipase A2 activity by Ca2+/calmodulin-dependent protein kinase II, cAMP-dependent protein kinase, and casein kinase II. 165 Apr 81

The activity of EF-2 was distinctly decreased after phosphorylation catalysed by a partly purified calmodulin and Ca2+ dependent protein kinase III. At the same time 32P from [gamma-32P]ATP was incorporated into EF-2 molecule. After dephosphorylation of EF-2 catalysed by alkaline phosphatase the activity of this factor was increased. This suggests that the phosphorylation-dephosphorylation of EF-2 is the regulatory process in the elongation step of the translation. Preliminary purification of the kinase III from rat liver resulted in 8-fold purified enzyme with a recovery of 60%.
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PMID:The effect of phosphorylation of the EF-2 isolated from rat liver cells on protein biosynthesis in vitro. 166 36


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