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)

The properties and developmental regulation of the protein phosphatases of Dictyostelium discoideum were examined. When crude extracts from vegetative cells were separated on a Mono Q column (FPLC) three protein phosphatase peaks, designated P1, P2 and P3 were found. When aggregation and culmination cells were examined only one protein phosphatase peak was observed. This corresponded to phosphatase P1 of vegetative cells. All three of the vegetative cell phosphatase were inhibited by heparin and mammalian phosphatase inhibitor-2, both of which are specific for type-1 protein phosphatases. Trifluoperazine, which inhibits type-2 protein phosphatases, had little effect on any peaks while levamisole, an alkaline phosphatase inhibitor, stimulated P2, slightly inhibited P3 and had no effect on P1. These results demonstrate the existence of two vegetative phase specific protein phosphatases in D. discoideum and one which occurs during all phases of the life cycle. The protein phosphatases isolated from vegetative cells all appear to be type-1 enzymes.
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PMID:Properties and developmental regulation of the protein phosphatases in Dictyostelium discoideum. 284 42

Stalk cell formation in low-cell-density monolayers of Dictyostelium discoideum, strain V12-M2, occurs following the sequential addition of cyclic AMP and the differentiation-inducing factor (DIF). Both cyclic AMP and DIF are essential for the appearance of the prestalk-specific isozyme alkaline phosphatase-II, which suggests that both factors are necessary for prestalk cell formation. The available evidence suggests that the cyclic AMP requirement for stalk cell formation is mediated through the cell surface cyclic AMP receptor. However, stalk cell formation is inhibited by caffeine and this inhibition is reversed by the cell-permeable analogue 8-Br-cyclic AMP, which suggests in addition a possible involvement for elevated intracellular cyclic AMP concentrations in stalk cell formation. During in vivo development cells first become independent of cyclic AMP at the tipped aggregate stage, but the acquisition of cyclic AMP independence is advanced by several hours when cells are incubated in the presence of cyclic AMP for 2 hours. Cells do not become independent of DIF until the culmination stage of development, which suggests the possibility that DIF is required for the conversion of prestalk cells to stalk cells. There is an absolute requirement for DIF for stalk cell formation in low-density monolayers of prestalk cells but only part of population exhibits a requirement for cyclic AMP, which suggests that the prestalk cell population consists of two distinct cell types. Stalk cell formation from prespore cells is totally dependent on both cyclic AMP and DIF.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stalk cell formation in monolayers of Dictyostelium discoideum V12-M2. 285 28

Cell fractionation studies have been performed, in order to obtain insight into the subcellular distribution of Dictyostelium adenylate cyclase and guanylate cyclase and also to provide a starting point for further study and isolation of these enzymes and their regulatory components. Adenylate cyclase and cAMP receptors were found in the same membrane fractions, but were distributed different from the plasma membrane marker alkaline phosphatase. Guanylate cyclase was partially soluble, partially particulate. In isopycnic gradients, particulate guanylate cyclase was present in other fractions than cAMP receptors and adenylate cyclase, but in similar ones to alkaline phosphatase. These observations are consistent with the hypothesis that cell-surface cAMP receptors and adenylate cyclase interact via a membrane-bound G-protein, whereas the receptors activate guanylate cyclase via a cytosolic factor. The adenylate cyclase activity in membranes obtained by sucrose gradient centrifugation was retained in the presence of various detergents, while with the same detergents the activity of particulate guanylate cyclase was lost. This adenylate cyclase was solubilized as assessed by gel filtration and centrifugation experiments, and it behaved heterogeneous in fractionation studies. In gel filtration, the major component eluted at a position corresponding to a Stokes radius of 4-7 nm. A purification of about 70-fold as compared to the cell homogenate was obtained by affinity chromatography of adenylate cyclase on ATP-Sepharose. We conclude that cell fractionation provides useful starting material for isolation and further study of Dictyostelium adenylate cyclase.
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PMID:Cell fractionation, detergent sensitivity and solubilization of Dictyostelium adenylate cyclase and guanylate cyclase. 288 13

During the aggregation and differentiation of amoebae of Dictyostelium discoideum, changes in free cytosolic Ca2+ appear to regulate a number of physiological processes. To understand the mechanisms regulating free intracellular Ca2+ in this organism, we have isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump. When homogenates of 2 h starved cells were fractionated on Percoll/KCl gradients, one peak of high-affinity Ca2+-pumping activity was detected. This activity was resolved from enzyme markers of the mitochondrion and the rough endoplasmic reticulum but it cosedimented with the plasma membrane marker, alkaline phosphatase. Further studies suggested that the pump was associated with 'inside-out' plasma membrane vesicles. Like plasma membrane Ca2+-transport ATPases from other systems, this isolated Ca2+ pump: (1) was Mg2+-dependent, (2) displayed a high specificity for ATP as an energy source, (3) exhibited a high affinity for free Ca2+ with a Km of 0.3 microM, and (4) was very sensitive to inhibition by vanadate (IC50 2 microM) but was unaffected by mitochondrial inhibitors, ouabain and Ca2+-channel blockers. Unlike plasma membrane Ca2+ pumps from most other systems, this enzyme appeared not to be regulated by calmodulin. During development, non-mitochondrial, vanadate-sensitive, high-affinity Ca2+-pumping activity in crude lysates remained relatively constant for at least 15 h. These observations suggest that this plasma membrane Ca2+ pump probably functions in Dictyostelium to maintain Ca2+ homeostasis by extruding free cytosolic Ca2+ from the cells.
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PMID:Isolation and characterization of a plasma membrane calcium pump from Dictyostelium discoideum. 296 24

Earlier reports suggested that the adenosine monophosphate (AMP)- and the p-nitrophenyl phosphate (pNPP)-hydrolyzing activities of Dictyostelium discoideum membrane preparations are due to different proteins. These results have been apparently contradicted by the recent purification to homogeneity of the two activities from culmination phase cells as a single protein [D. R. Armant and C. L. Rutherford (1981) J. Biol. Chem. 256, 12710-12718]. Results presented here from studies on the activities of vegetative cells support the concept of a single protein. Nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Triton X-100 extracts of cell membrane preparations of D. discoideum showed identical migration of pNPPase and AMPase activities. Furthermore, the previously reported different pH optima of the two activities was due to the fact that pH optima are dependent upon the substrate concentration, and the selective solubilization of AMPase from membrane preparations by phospholipase C can probably be accounted for by the finding that phospholipase C preparations from the same commercial source contain 5'-nucleotidase activity. Moreover, there are alterations in the Km and the stability of both AMPase and pNPPase in a strain with a mutationally altered alkaline phosphatase, further supporting the concept that the two activities are due to a single protein. Both substrates serve as transphosphorylation donors demonstrating that the enzyme activity is mechanistically an alkaline phosphatase.
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PMID:The membrane-bound alkaline phosphatase and 5'-nucleotidase activities of vegetative cells of Dictyostelium discoideum. 298 13

Mouse P388D1 macrophages target newly synthesized acid hydrolases to lysosomes in spite of their lack of the 215-kDa mannose 6-phosphate (Man-6-P) receptor. We now report that these cells contain a membrane-associated Man-6-P receptor that is distinct from the previously described receptor. The new receptor binds lysosomal enzymes containing phosphomannosyl residues. This binding is inhibited by Man-6-P or by pretreatment of the lysosomal enzymes with alkaline phosphatase. Lysosomal enzyme binding occurs at neutral pH and dissociation of the bound ligand occurs at low pH values comparable to those found within endosomes or lysosomes. The new receptor differs from the 215-kDa Man-6-P receptor in two ways. It has an absolute requirement for divalent cations and is unable to bind Dictyostelium discoideum lysosomal enzymes, which contain methylphosphomannosyl residues rather than the usual phosphomannosyl monoesters. Based on the difference in cation requirement, we suggest that the 215-kDa receptor be referred to as Man-6-P receptor CI (cation independent) and the new receptor as Man-6-P receptor CD (cation dependent). We conclude that the Man-6-P receptor CD functions in the targeting of newly synthesized acid hydrolases to lysosomes in P388D1 macrophages.
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PMID:Lysosomal enzyme binding to mouse P388D1 macrophage membranes lacking the 215-kDa mannose 6-phosphate receptor: evidence for the existence of a second mannose 6-phosphate receptor. 316 44

A conjugate of hippuryllysine (HP) and adenylic acid was synthesized and purified. The structure of the conjugate, hippuryllysyl(N-epsilon-5'-phospho)adenosine (HLAMP) was established using 31P nuclear magnetic resonance, UV spectroscopy, acid/base lability, and enzyme digestion with AMP deaminase, alkaline phosphatase, 5'-nucleotidase, and a phosphoamidase activity recently identified in Dictyostelium discoideum. The results indicate that HLAMP contains a phosphoamide bond between the phosphate of AMP and the epsilon amino group of HL. Employing a microdroplet assay to assess chemotactic activity, HLAMP was found to be a potent chemoattractant of 7-h developing amoebae of D. discoideum. Other conjugates, including lysine-AMP (LAMP), tuftsin-AMP (TAMP) and avidin-AMP (AVAMP), as well as the degradation products of HLAMP (HL, AMP, and lysine) exhibited no chemotactic activity. The molecular structure of HLAMP is compared to that of other known chemoattractants of the cellular slime molds, and possible chemotactic receptors for HLAMP are considered.
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PMID:HLAMP--a conjugate of hippuryllysine and AMP which contains a phosphoamide bond--stimulates chemotaxis in Dictyostelium discoideum. 344 32

We demonstrated that alkaline phosphatase was localized on the cell membrane of Dictyostelium discoideum amebae and on isolated plasma membranes. The enzyme activity was specifically inhibited by 0.01 M KCN or cysteine. The same method could also be applied to baker's yeast and MDCK cells (dog kidney cells in vitro).
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PMID:Cytochemical localization of alkaline phosphatase in the cell membrane of Dictyostelium discoideum amebae. 404 10

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N(2) and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [(125)I]iodide. 5'-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH-cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH-cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.
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PMID:The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum. 415 70

1. The specific activities of beta-N-acetylglucosaminidase, acid phosphatase, alpha-mannosidase, beta-glucosidase, UDP-glucose pyrophosphorylase and alkaline phosphatase have been determined in myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 grown on different media and in different phases of the growth cycle. 2. Variations in enzymic composition occur with changes in growth medium and phase of the growth cycle. 3. The intracellular location of the enzymes studied has been determined. 4. Two enzymes, beta-N-acetylglucosaminidase and alpha-mannosidase, are not only synthesized preferentially as the myxamoebae enter the stationary phase of growth but they are also excreted. The excretion process appears to be specific, because other enzymes that occur in the same intracellular fraction are not excreted.
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PMID:Enzyme synthesis in myxamoebae of the cellular slime mould Dictyostelium discoideum during growth in axenic culture. 467 68


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