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)

Cytoplasmic granules obtained from toad urinary bladder epithelial cells were brought to buoyancy in a linear sucrose gradient. The gradient was loaded either with untreated cytoplasmic granules, or with granules treated with Na pyrophosphate (PPi), with digitonin, or with PPi and digitonin in succession. The following enzymes were assayed in the gradient subfractions: oligomycin-insensitive Mg++-ATPase, alkaline phosphodiesterase I, alkaline phosphatase, acid N-acetyl-beta-glucosaminidase, cytochrome oxidase, nucleoside diphosphatase (substrate, ADP), aminopeptidase (substrate, leucyl-beta-naphthylamide), and mannosyltransferase (acceptor, dolichylphosphate). Comparison of the density distributions of enzymes in untreated and treated preparations led to the characterization of 4 distinct subcellular entities. In agreement with the properties of mitochondria from other cell types, cytochrome oxidase buoys at 1.18 within a narrow density range and its behavior is not significantly altered by PPi or digitonin. Under all conditions, acid N-acetyl-beta-glucosaminidase is recovered over a broad density range in the lower part of the gradient and appears as a qualified lysosomal marker. Mg++-ATPase, alkaline phosphodiesterase I, and alkaline phosphatase belong to a group with the distinguishing features of a low equilibrium density in native cytoplasmic granules and a marked shift (+0.03 density units) after digitonin treatment. Such properties are typical of the plasma membranes. Part of the aminopeptidase activity probably also belongs to plasma membrane-derived elements. Minor differences between alkaline phosphatase and the other 2 members of that group make it possible that their distribution domains in the membrane do not overlap or coincide.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Subcellular fractionation of epithelial cells from toad urinary bladder. 2. Isopycnic centrifugation and effect of density perturbants. 255 74

Retroplacental blood flow requires inhibition of coagulation in the absence of an endothelial lining. We confirmed that trophoblast releases an inhibitor of platelet aggregation which functions via degradation of adenosine diphosphate. This inhibitor appears to be deficient in some pregnancies with abruptio placentae and intrauterine growth retardation. Unimpeded retroplacental blood flow may depend upon the local inhibition of platelet aggregation. Placental tissue contains an inhibitor of platelet aggregation which appears to be an adenosine diphosphatase distinct from heat-stable alkaline phosphatase. Placental tissue from patients with abruptio placentae contains abnormally low amounts of this enzyme.
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PMID:Platelet inhibitory activity in placentas from normal and abnormal pregnancies. 281 99

Inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) is an important second-messenger molecule that mobilizes Ca2+ from intracellular stores in response to the occupancy of receptor by various Ca2+-mobilizing agonists. The fate of Ins-1,4,5-P3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P3 to Ins-1,3,4,5-P4, whereas the latter forms Ins-1,4-P2. Recent studies suggest that Ins-1,3,4,5-P4 might modulate the entry of Ca2+ from an extracellular source. In the current report, we describe the partial purification of the 3-kinase [approximately 400-fold purified, specific activity = 0.12 mumol/(min.mg)] from the cytosolic fraction of bovine brain and studies of its catalytic properties. We found that the 3-kinase activity is significantly activated by the Ca2+/calmodulin complex. Therefore, we propose that Ca2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P3 forms a complex with calmodulin, and that the Ca2+/calmodulin complex stimulates the conversion of Ins-1,4,5-P3, an intracellular Ca2+ mobilizer, to Ins-1,3,4,5-P4, an extracellular Ca2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3-32P]Ins-1,3,4,5-P4 and [gamma-32P]ATP by thin-layer chromatography. Using this new assay method, we evaluated kinetic parameters (Km for ATP = 40 microM, Km for Ins-1,4,5-P3 = 0.7 microM, Ki for ADP = 12 microM) and divalent cation specificity (Mg2+ much greater than Mn2+ greater than Ca2+) for the 3-kinase. Studies with various inositol polyphosphates indicate that the substrate-binding site is quite specific to Ins-1,4,5-P3. Nevertheless, Ins-2,4,5-P3 could be phosphorylated at a velocity approximately 1/20-1/30 that of Ins-1,4,5-P3.
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PMID:Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin. 282 70

Human neutrophils stimulated with immune complexes in the presence of platelets show enhanced superoxide anion (O2-.) responses that are proportional to the amount of agonist present and the number of platelets added. Platelet related enhancement of O2-. responses also occurs with the neutrophil agonists phorbol ester, formyl chemotactic peptide and zymosan particles. Pretreatment of platelets with cycloheximide does not alter their ability to enhance O2-. responses of neutrophils. In parallel with platelet-related enhancement of O2-. responses of immune complex-stimulated neutrophils, secretory release of myeloperoxidase is also increased. The platelet effects on O2-. responses can be reproduced with platelet lysates or with supernatant fluids which have been obtained from thrombin or immune complex-stimulated platelets and are rich in ATP and ADP content. Solutions containing ATP and ADP in amounts similar to those found in supernatant fluids of activated platelets reproduce the enhancement of O2-. responses in N-formyl methionyl leucyl phenylalanine (FMLP) or immune complex-activated neutrophils. The platelet factor responsible for the effects of neutrophils is heat-stable, elutes in gel sieving chromatography near the position of phenol red and does not, in the absence of a neutrophil agonist, trigger an O2-. response. With formyl peptide-stimulated neutrophils, ATP and ADP enhance O2-. responses while the responses are depressed by addition of AMP or adenosine. In immune complex-stimulated neutrophils, adenosine and all adenine nucleotides enhance the O2-. responses. Taking advantage of this information, treatment of ATP or of supernatant fluids from thrombin-stimulated platelets with alkaline phosphatase (resulting in formation of adenosine) converts the O2-. enhancing activity for formyl peptide-activated neutrophils into an inhibitory activity. In contrast, using immune complex-activated neutrophils, similar manipulations of ATP or supernatant fluids from stimulated platelets result only in enhanced O2-. responses. These data support the conclusion that the platelet-derived factor responsible for enhanced O2-. responses in neutrophils is ATP/ADP. In FMLP stimulated neutrophils, the presence of ATP or ADP leads to enhanced increases in intracellular levels of Ca++ as determined by the fura-2 probe, while the presence of AMP or adenosine results in inhibition of the increases in FMLP induced elevations in cytosolic Ca++. These data demonstrate a direct relationship between effects of adenine compounds on FMLP induced changes in cytosolic Ca++ and the associated O2-. responses.
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PMID:Platelet enhancement of O2-. responses in stimulated human neutrophils. Identification of platelet factor as adenine nucleotide. 282 82

Retinoic acid (RA) inhibits the increases in alkaline phosphatase (AP) and hormone-stimulated adenylate cyclase that accompany the growth of ROS 17/2.8 osteosarcoma cells in culture. The RA effects were first detected 2 days after initiation of treatment and were dose dependent, with an EC50 of 100 nM. The reduction in the hormone-responsive adenylate cyclase activity was associated with lower levels of beta-catecholamine receptors, without a change in apparent receptor affinity and with lower levels of the GTP-binding proteins Gs and Gi, visualized by NAD-dependent [32P]ADP ribosylation. The reduction in AP was correlated with a decrease in the steady state level of AP mRNA. RA had no effect on cell proliferation or saturation density. Retinoids thus inhibit the same features that are promoted by glucocorticoids in ROS 17/2.8 cells. These features seem to be subject to coordinate regulation, probably at the pretranslational level.
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PMID:Effects of retinoic acid on alkaline phosphatase messenger ribonucleic acid, catecholamine receptors, and G proteins in ROS 17/2.8 cells. 282 98

The regulatory site peptide sequence of phosphorylated inactive pyruvate, orthophosphate dikinase from maize leaf tissue was determined by automated Edman degradation analysis of 32P-labeled peptides purified by reversed-phase high performance liquid chromatography. The overlapping phosphopeptides were products of a digestion of the [beta-32P]ADP-inactivated dikinase with either trypsin or Pronase E. The sequence is Thr-Glu-Arg-Gly-Gly-Met-Thr(P)-Ser-His-Ala-Ala-Val-Val-Ala-Arg. The phosphothreonine residue, which appeared as either an anomalous proline or an unidentifiable phenylthiohydantoin derivative during sequencing, was verified by two-dimensional phosphoamino acid analysis of the phosphopeptides and by resequencing the tryptic peptide after dephosphorylation with exogenous alkaline phosphatase. This sequence, starting at position 4, is completely homologous to the previously published sequence of the tryptic dodecapeptide harboring the catalytically essential (phospho)histidyl residue in the active-site domain of the dikinase from the nonphotosynthetic bacterium, Bacteroides symbiosus (Goss, N.H., Evans, C.T., and Wood, H.G. (1980) Biochemistry 19, 5805-5809). These comparative results indicate that the regulatory phosphothreonine causing complete inactivation of maize leaf dikinase is separated from the critical active-site (phospho)histidine by just one intervening residue in the primary sequence.
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PMID:Sequence of the phosphothreonyl regulatory site peptide from inactive maize leaf pyruvate, orthophosphate dikinase. 283 85

An enzyme with FAD pyrophosphatase activity was extracted from human placental syncytiotrophoblast microvilli and purified to near-homogeneity. The enzyme has been identified as 5'-nucleotidase by several criteria. Throughout purification, parallel increases in the specific activities of FAD pyrophosphatase and AMP phosphatase were observed. The enzyme was a glycoprotein with a subunit molecular weight of 74,000. EDTA treatment resulted in a marked decline in both activities, and restoration of FAD pyrophosphatase activity but not 5'-nucleotidase activity was accomplished by the addition of Co2+ or, to a lesser extent, Mn2+. The substrate specificity of the 5'-nucleotidase activity that we observed agreed closely with the results of others. The pyrophosphatase activity was relatively specific for FAD. ADP, ATP, NAD(H), and FMN were not hydrolyzed, and ADP strongly inhibited both activities. For FAD pyrophosphatase activity, a Km of 1.2 x 10(-5) M and a Vmax of 1.1 mumol/min/mg protein were determined in assays performed in the presence of Co2+. In the absence of added Co2+, the Vmax declined but the Km was unchanged. For 5'-nucleotidase (AMP as substrate) the Km was 4.1 x 10(-5) M and the Vmax 109 mumol/min/mg protein. Hydrolysis of FMN to riboflavin was observed in partially purified detergent extracts of microvilli that contained alkaline phosphatase activity and lacked FAD pyrophosphatase and 5'-nucleotidase activity. The presence of both FAD pyrophosphatase and FMN phosphatase activities in syncytiotrophoblast microvilli supports the view that the placental uptake of vitamin B2 involves the hydrolysis of FAD and FMN to riboflavin which is then absorbed, a sequence postulated for intestinal absorption and liver uptake.
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PMID:5'-Nucleotidase of human placental trophoblastic microvilli possesses cobalt-stimulated FAD pyrophosphatase activity. 284 89

H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase. The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference. Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9. ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM. The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole. It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein. These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase. The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S. cerevisiae.
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PMID:Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. 285 69

To investigate the effect of moderate alcohol consumption on blood constituents related to cardiovascular disease, 12 male volunteers consumed (instead of their usual alcoholic drinks) four different standardized amounts of red wine in addition to their habitual diet. Each dose was given to the subjects during a period of 5 weeks in a randomized order, all subjects receiving the four doses. They consisted of 0, 2, and 4 glasses/d, providing 0, 23, and 46 g alcohol/d as well as in "binge drinking" (14 glasses in the weekend, comparable to an average of 2 glasses/d). The results showed a clear dose-related response to the drinking for several blood constituents. Most marked was a decrease in the tissue-type plasminogen activator activity and to a lesser degree an increase in plasminogen levels. Collagen-induced platelet aggregation was reduced, affecting all parameters measured. Levels of HDL3-cholesterol, gammaglutamyltransferase, and urate showed a small but significant increase. No change was noted in the levels of alkaline phosphatase, alanine-aminotransferase, aspartate-aminotransferase, bile acids, folate, fibrinogen, the ADP-induced platelet aggregation, platelet secretion, or in hematologic values. The results are only partially in accordance with the presumed protective action of moderate drinking on the cardiovascular system and show a stronger response to the consumption of alcohol in coagulation and fibrinolysis factors than in blood lipids.
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PMID:Effects of moderate alcohol consumption on platelet aggregation, fibrinolysis, and blood lipids. 288 51

Isolation procedures employed by various laboratories to obtain cerebral microvessels generally utilize meshes to sieve and collect the microvessels from homogenized brain. This is followed in some cases by further purification using density gradients of Percoll or sucrose, or albumin flotation. We have evaluated microvessels prepared by these methods in terms of ATP content and ATP/ADP ratio, which reflect the cellular energy state, and enrichment of the marker enzymes, alkaline phosphatase and gamma-glutamyltransferase. Albumin flotation generally increased the enrichment of marker enzymes; however, preparations using albumin flotation or a Percoll gradient exhibited considerable variability in ATP content and ATP/ADP ratio with the mean ATP/ADP ratio significantly lower than that observed in microvessels isolated by sieving through meshes. More uniformly high values for both ATP (approximately 1.6 nmole ATP/mg protein) and the ATP/ADP ratio (approximately 2.3) were obtained with meshes alone. Use of a sucrose gradient consistently resulted in preparations with a much lower ATP content and ATP/ADP ratio, compared with preparations obtained with the other methods. Values using the other methods were higher than those previously reported, yet were still lower than the ATP content of about 23 and ATP/ADP ratios of 18 and 7 we found in cultured microvascular endothelium and pericyte, respectively. These low values were not improved by supplying additional fuel to the microvessels during isolation, suggesting they were not the result of fuel deprivations during isolation. Despite the probable damage incurred during isolation, microvessel preparations are a useful in vitro model in which fuel metabolism appears to reflect the prior hormonal/nutritional state of donor animals. However, our data indicate the advisability of measurements of ATP content and ATP/ADP ratio for quality control of preparations used for metabolic studies, especially after Percoll density gradient or albumin flotation steps.
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PMID:Energy state of bovine cerebral microvessels: comparison of isolation methods. 289 72


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