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)

Short-term treatment of rat submandibular tissues with 10 microM isoproterenol (IPR) resulted in reduction of mucin secretion in response to the agonist during further incubation, and in increases in EC50 values. This IPR-induced reduction of secretion was coupled with selective decreases in the number of beta-adrenoceptors in the tissues and in their affinity for agonists, as assessed by measurement of the specific binding of [3H]dihydroalprenolol. Treatment of the tissues with IPR caused a 30% decrease in IPR-stimulated adenylate cyclase activity and a 25% increase in the GTP binding capacity of inhibitory G proteins (Gi proteins). This IPR treatment triggered a 60% increase in the ability of pertussis toxin (IAP) to catalyze ADP-ribosylation of Gi proteins in the tissue membranes. Enhanced function of stimulatory G proteins (Gs proteins) was observed only during the first incubation of the tissues with IPR. The IAP-catalyzed ADP-ribosylation of Gi proteins in tissues treated with IPR was decreased by prior treatment with cyclic AMP dependent protein kinase, but was increased markedly by prior treatment with alkaline phosphatase. Neither IPR-induced desensitization of protein secretion nor increase in the IAP-catalyzed ADP-ribosylation of Gi proteins was observed in the tissues pretreated with 0.25 microM okadaic acid. These findings suggest that the regulation of Gi protein phosphorylation plays an important role in the IPR-induced heterologous desensitization of mucin secretion from rat submandibular glands.
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PMID:Mechanism of isoproterenol-induced heterologous desensitization of mucin secretion from rat submandibular glands. Regulation of phosphorylation of Gi proteins controls the cell response to the subsequent stimulation. 769 46

Antibodies against the holo ecto-adenosinetriphosphatase (ATPase) of rat liver and antibodies against COOH-terminal peptides of the long isoform of this enzyme reacted in Western blots with a 105-kDa band from small intestinal brush-border membranes. Indirect immunofluorescence revealed reactive proteins predominantly at the apical surface of enterocytes with some staining of basolateral membranes and of vascular endothelium. Similar results were obtained with monoclonal antibodies against HA4, a protein from rat liver closely related to the ecto-ATPase. Since these results suggested the presence of an ecto-ATPase, ATP hydrolysis was studied in intact, right-side-out brush-border membrane vesicles. Nearly half of ATP hydrolysis was caused by alkaline phosphatase (AP). Besides purine and pyrimidine trinucleotides, AP also hydrolyzed ADP, AMP, pyrophosphate, and 4-nitrophenylphosphate. Inactivation of AP by cleavage of its membrane anchor and by removal of the Zn2+ necessary for its function left the ecto-ATPase that was activated by Ca2+ and Mg2+ and hydrolyzed purine and pyrimidine trinucleotides and dinucleotides, but not AMP, pyrophosphate, and 4-nitrophenylphosphate. These features are characteristic of an ATP diphosphohydrolase (EC 3.6.1.5, also called apyrase). The physiological role of the small intestinal ecto-ATPase may be the degradation of nutrient nucleotides.
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PMID:Ecto-adenosinetriphosphatase in rat small intestinal brush-border membranes. 773 91

Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (Pi) from the breakdown of extracellular ATP. Enzyme activity involved Ca2+/Mg(2+)-dependent and Ca2+/Mg(2+)-independent dephosphorylation of ATP. Ca2+/Mg(2+)-dependent ecto-ATPase was active over a limited range of 0.01-1.0 mM ATP, while Ca2+/Mg(2+)-independent ATPase activity was active over a range of 0.1-30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0-9.0), but increased in a relatively linear manner with: (1) time of reaction (0-90 min), (2) number of cells (1-20 oocytes), and (3) temperature (10-37 degrees C). Ecto-ATPase activity was unaffected by ouabain (100 microM), sodium azide (100 microM), and oligomycin (5 micrograms/ml) (as inhibitors of endo-ATPases) and beta-glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methylthioATP>ADP, 2-methylthioADP, AMP>>alpha, beta-methylene ATP, beta, gamma-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P2-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido)napthalene-1,3,5-trisul phonic acid), 100 microM] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca2+/Mg(2+)-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characteristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown. 892 22

cAMP is commonly measured using either immunoassay or high-performance liquid chromatography. The current methods are sensitive but may lack versatility and be expensive; also, radioactivity is potentially harmful to the operator and environment. Given these concerns, we developed a highly sensitive enzymatic fluorometric assay for cAMP. The method consists of five steps: (1) destruction of interfering compounds with apyrase, 5' nucleotidase, adenosine deaminase, and alkaline phosphatase; (2) conversion of cAMP to AMP; (3) conversion of AMP to ATP; (4) amplification of ATP by ATP-ADP cycling; and (5) fluorometric measurement of resultant NADPH. cAMP was measured in male Sprague Dawley rats anesthetized with pentobarbital. Stimulated rats (n = 4) received isoproterenol (16 micrograms/kg, s.q.) and aminophylline (20 mg/kg, s.q.), whereas controls (n = 4) received no additional drug. With the enzymatic fluorometric assay, cAMP content in heart, liver, and kidney (pmol/mg wet wt, mean +/- SEM) was 0.34 +/- 0.03, 0.33 +/- 0.03, and 0.92 +/- 0.11 in the control group and 0.77 +/- 0.10, 0.66 +/- 0.04, and 1.53 +/- 0.12 in the stimulated group, respectively. The total assay duration including sample reading procedure varied at 4.5-9.5 hr, depending on its sensitivity. cAMP from the same samples was measured using a commercially available enzyme immunoassay kit and was found to be very similar to the enzymatic fluorometric assay. We conclude that this new assay is sensitive, safe, versatile, and inexpensive and can be used to measure cAMP in multiple types of tissue, including biopsy samples weighing < 200 micrograms.
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PMID:Enzymatic fluorometric assay for tissue cAMP. 786 85

The interaction of myosin subfragment 1 isoenzyme A2 (S1A2) with Mg(2+)-G-actin was studied. Polarization titrations of 1,5-IAEDANS-Mg(2+)-G-actin and of epsilon ATP-Mg(2+)-G-actin with S1A2 provided evidence that, similar to Ca(2+)-G-actin, the proteins form a tight binary complex. Significant amounts of oligomeric forms of actin in the presence and absence of S1 were not detected. The effect of S1A2 on the rates of nucleotide and metal dissociation and hydrolysis from Mg(2+)-actin was measured. The hydrolysis rate for [gamma-32P]ATP-actin in the G-acto-S1A2 complex (k- = 0.016 s-1) was faster than the rate of 32P liberation from the complex (k- = 0.004 s-1), obtained by measuring the liberation of [32P]orthophosphate from [alpha-32P]ATP-actin in the presence of a large excess of alkaline phosphatase. This indicates that most of actin's ATP was hydrolyzed before it was released to solution and that the dissociating nucleotide was ADP, for which the dissociation rate is higher than that for ATP. In agreement with this mechanism, S1A2 accelerated the dissociation of epsilon ATP but inhibited the dissociation of epsilon ADP from the complex. The activation of actin's ATPase is specific for Mg(2+)-G-actin and does not occur in Ca(2+)-G-actin. The effect of deoxyribonuclease I on the rates of nucleotide dissociation and hydrolysis was examined.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Myosin subfragment 1 activates ATP hydrolysis on Mg(2+)-G-actin. 791 68

Human placental chorioepithelial brush border membrane, which is in direct contact with maternal blood flow, has platelet aggregation inhibiting activity. In the present study, the mechanism of this action has been examined in relation to ADP (adenosine diphosphate) degrading activity and alkaline phosphatase activity of brush border membrane vesicles (BBMV). BBMV prepared from human early and term placental villi, inhibited platelet aggregation induced by ADP. BBMV had potent ADP degrading (ADPase) activity. ADP was quickly degraded by BBMV. ADP degrading activity of BBMV was not so different between early and term placenta. Alkaline phosphatase activity of late placental BBMV was about three times greater than that of early placental BBMV. On the other hand, ADP degrading activity of late placental BBMV was almost the same as that of early placental BBMV. Inhibiting activity of platelet aggregation induced by ADP and ADP degrading activity of BBMV, were not inhibited by levamisole (alkaline phosphatase inhibitor).
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PMID:Platelet aggregation inhibiting activity of human placental chorioepithelial brush border membrane vesicles--the role of alkaline phosphatase. 806 52

Human placental mitochondria prepared by a new isolation procedure exhibit low but well coupled rates of state 3 respiration with different substrates (succinate: 32.3 nmol O2/mg/min, RCI = 4.4; pyruvate: 12.6 nmol O2/mg/min, RCI R = 4.2; palmitoylcarnitine: 16.6 nmol O2/mg/min, RCI R = 4.9). The addition of the uncoupler FCCP increased the respiratory rates (succinate: 40.7 nmol O2/mg/min; pyruvate: 21.2 nmol O2/mg/min: palmitoylcarnitine: 25.4 nmol O2/mg/min). The low respiratory rates correlate well with a low capacity of the respiratory chain as shown by the specific contents of cytochrome c (0.15 nmol/mg), cytochrome b (0.19 nmol/mg) and cytochrome oxidase (0.14 nmol/mg) as well as with the low content of adenine nucleotides (2.71 nmol/mg). These data together with the finding of high activities of alkaline phosphatase (2.2 U/mg) support the view that human placental mitochondria are contaminated with nonmitochondrial membranes. Since it was not possible to obtain functionally intact mitochondria with negligible activities of alkaline phosphatase the influence of this enzyme on the extramitochondrial adenine nucleotide turnover was investigated. Alkaline phosphatase splits phosphate from ATP, ADP and AMP with different rates resulting in an intermediate accumulation of AMP. Mitochondrial adenylate kinase (0.16 U/mg) regenerated ADP from AMP and ATP resulting in drastically decreased ADP/O ratios and prolonged state 3 respirations. Inhibiting the adenylate kinase with diadenosine pentaphosphate the ADP regeneration from AMP and ATP was suppressed which, in turn, enhanced the ADP/O ratios. In the absence of magnesium ions, if both the alkaline phosphatase and the adenylate kinase are inhibited normal ADP/O ratios and state 3-state 4 transitions can be observed. Under these conditions, human placental mitochondria showed normal properties comparable to those of mitochondria from other tissues with the only exception of low specific activities.
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PMID:Unusual properties of mitochondria from the human term placenta are caused by alkaline phosphatase. 806 53

In the present report we describe an apyrase (ATP diphosphohydrolase, EC 3.6.1.5) in rat blood platelets. The enzyme hydrolyses almost identically quite different nucleoside di- and triphosphates. The calcium dependence and pH requirement were the same for the hydrolysis of ATP and ADP and the apparent Km values were similar for both Ca(2+)-ATP and Ca(2+)-ADP as substrates. Ca(2+)-ATP and Ca(2+)-ADP hydrolysis could not be attributed to the combined action of different enzymes because adenylate kinase, inorganic pyrophosphatase and nonspecific phosphatases were not detected under our assay conditions. The Ca(2+)-ATPase and Ca(2+)-ADPase activity was insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors, thus excluding these enzymes as contaminants. The results demonstrate that rat blood platelets contain an ATP diphosphohydrolase involved in the hydrolysis of ATP and ADP which are vasoactive and platelet active adenine nucleotides.
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PMID:Characterization of an ATP diphosphohydrolase activity (APYRASE, EC 3.6.1.5) in rat blood platelets. 817 26

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins, which, like other members of the ras superfamily, are activated by exchanging bound GDP for GTP and inactivated through hydrolysis of the gamma-phosphate of bound GTP to form GDP in a highly regulated cycle. ARF 6, a class III ARF, was expressed in Escherichia coli with its amino terminus fused to maltose-binding protein. Following release from maltose-binding protein, recombinant ARF 6 (rARF 6) exhibited maximal activity with or without GTP. Such constitutive activation was due to the predominance of ARF-GTP over ARF-GDP, as demonstrated by nucleotide analysis. rARF 6 expressed in E. coli without amino-terminal extension was bound primarily to GDP and exhibited typical GTP-dependent activity. After release from maltose-binding protein, rARF 6-GTP was stable; only a fraction of the nucleotide was removed using EDTA, whereas urea denaturation restored complete GTP dependence. [alpha-32P]GTP bound to rARF 6 was in part protected from hydrolysis by alkaline phosphatase and resulted in the formation of [alpha-32P]GTP, -GDP, and -GMP, whereas unbound nucleotide was completely hydrolyzed to guanosine. Thus, amino-terminal extension of rARF 6, by maltose-binding protein, promoted the formation of a constitutively activated GTP-bound species. By analysis of this species, we confirmed that rARF 6 lacks the intrinsic ability to hydrolyze bound GTP and speculate that maltose-binding protein may inhibit hydrolysis by extrinsic factors.
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PMID:Isolation of recombinant ADP-ribosylation factor 6, an approximately 20-kDa guanine nucleotide-binding protein, in an activated GTP-bound state. 819 4

Specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study the involvement of phosphorylation in the regulation of the low-molecular-mass GTP-binding protein Rho. Dephosphorylation of CHO cell extracts by alkaline phosphatase treatment resulted in a 80-90% reduction in the C3-catalysed [32P]ADP-ribosylation of Rho proteins in both cytosolic and membrane fractions. Similar results were obtained after dephosphorylation with protein phosphatase type-1 from bovine retina, whereas type-2B and type-2C phosphatases had no effect on the level of subsequent [32P]ADP-ribosylation of Rho by C3. Incubation of CHO cell lysate under phosphorylation conditions increased the subsequent C3-mediated [32P]ADP-ribosylation of Rho proteins. The protein kinase inhibitors H7 and H9 had no effect on [32P]ADP-ribosylation at concentrations which are specific for inhibition of protein kinase A or C. Recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was phosphorylated by protein kinase A; however, the phosphorylation had no stimulatory effect on the ADP-ribosylation of GST-RhoA by C3. An approx. 48 kDa phosphoprotein was identified which bound specifically to recombinant GST-RhoA fusion protein. By gel-permeation chromatography, Rho-containing complexes of approx. 50 kDa and 130-170 kDa were detected. The ADP-ribosylation of Rho in the 130-170 kDa complex was reduced by alkaline phosphatase pretreatment. The data suggest that Rho activity is influenced by phosphorylation of Rho-associated regulatory factors. Phosphorylation/dephosphorylation of these Rho-regulating factors appears to alter the ability of Rho to serve as a substrate for C3-induced [32P]ADP-ribosylation.
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PMID:ADP-ribosylation of Rho proteins by Clostridium botulinum exoenzyme C3 is influenced by phosphorylation of Rho-associated factors. 819 24


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