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)

Alkaline phosphatase in a wide range of tissues has been shown to be anchored in the membrane by a specific interaction with the polar head group of phosphatidylinositol. It has previously been suggested that the production of low Mr alkaline phosphatase during the commonly used butanol extraction procedure may result from the activation of an endogenous phosphoinositide-specific phospholipase C which removes the 1,2-diacylglycerol responsible for membrane anchoring. This conversion process was investigated in greater detail with human placenta used as the source of alkaline phosphatase. Mr and hydrophobicity of the alkaline phosphatase were determined by gel filtration on TSK-250 and partitioning in Triton X-114, respectively. Alkaline phosphatase extracted from human placental particulate fraction with butanol at pH 5.4 or released by incubation with Staphylococcus aureus phosphatidylinositol-specific phospholipase C produced a form of alkaline phosphatase of Mr approx. 170,000 and relatively low hydrophobicity. By contrast, the butanol extract prepared at pH 8.3 was an aggregated form of Mr approx. 600,000 and was relatively hydrophobic. The effect of a variety of inhibitors and activators on the amount of low Mr alkaline phosphatase produced during butanol extraction revealed that it was a Ca2+- and thiol-dependent process. Proteinase inhibitors had no effect. [3H]Phosphatidylinositol hydrolysis by the particulate fraction, unlike low Mr alkaline phosphatase production, was relatively sensitive to heat inactivation, indicating that the phosphoinositide-specific phospholipases C from cytosol and lysosomes were unlikely to be responsible for conversion. A butanol-stimulated activity which removed the [3H]myristic acid from the variant surface glycoprotein ( [3H]mfVSG) of Trypanosoma brucei was detectable in the human placental particulate fraction. Since this activity was acid active, Ca2+- and thiol-dependent and relatively heat stable, it may be the same as that responsible for production of low Mr alkaline phosphatase. The only 3H-labelled product identified was phosphatidic acid, suggesting that the [3H]mfVSG-cleaving activity is a phospholipase D. These data strongly support the proposal that production of low Mr alkaline phosphatase during butanol extraction is an autolytic process occurring as the result of an endogenous phospholipase. However, they also suggest that the lysosomal and cytosolic phosphoinositide-specific phospholipases C that have previously been described in many mammalian tissues are not responsible for this process.
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PMID:Conversion of human placental alkaline phosphatase from a high Mr form to a low Mr form during butanol extraction. An investigation of the role of endogenous phosphoinositide-specific phospholipases. 302 77

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.
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PMID:Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. 312 81

Two enzymes, alkaline phosphatase and acetylcholinesterase (AChE), have been shown previously to be components of the surface of the trematode parasite Schistosoma mansoni. In this study we report that both these enzymes and other serine hydrolases are susceptible to release from the S. mansoni tegumental membrane by a phosphatidylinositol-specific phospholipase C (PIPLC) of bacterial origin. These data suggest that AChE and alkaline phosphatase of S. mansoni, as in higher organisms, are anchored to the membrane via covalently attached phosphatidylinositol. The release of AChE from the vesicular fraction of the parasite with PIPLC occurs in a concentration-dependent manner. Sucrose gradient centrifugation of the PIPLC-released AChE showed a single 8.3 S molecular form, similar to that observed for AChE solubilized by Triton X-100. PIPLC removed large amounts of AChE from the surface of intact schistosomula in culture, with no impairment of the viability of the parasite. In this case, an increase in the overall levels of AChE in the intact parasite was observed after addition of PIPLC.
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PMID:Acetylcholinesterase in Schistosoma mansoni is anchored to the membrane via covalently attached phosphatidylinositol. 313 66

The 200-kD subunit of neurofilaments (NF-H) functions as a cross-bridge between neurofilaments and the neuronal cytoskeleton. In this study, four phosphorylated NF-H variants were identified as major constituents of axons from a single neuron type, the retinal ganglion cell, and were shown to have characteristics with different functional implications. We resolved four major Coomassie Blue-stained proteins with apparent molecular masses of 197, 200, 205, and 210 kD on high resolution one-dimensional SDS-polyacrylamide gels of mouse optic axons (optic nerve and optic tract). Proteins with the same electrophoretic mobilities were radiolabeled within retinal ganglion cells in vivo after injecting mice intravitreally with [35S]methionine or [3H]proline. Extraction of the radiolabeled protein fraction with 1% Triton X-100 distinguished four insoluble polypeptides (P197, P200, P205, P210) with expected characteristics of NF-H from two soluble neuronal polypeptides (S197, S200) with few properties of neurofilament proteins. The four Triton-insoluble polypeptides displayed greater than 90% structural homology by two-dimensional alpha-chymotryptic iodopeptide map analysis and cross-reacted with four different monoclonal and polyclonal antibodies to NF-H by immunoblot analysis. Each of these four polypeptides advanced along axons primarily in the Group V (SCa) phase of axoplasmic transport. By contrast, the two Triton-soluble polypeptides displayed only a minor degree of alpha-chymotryptic peptide homology with the Triton-insoluble NF-H forms, did not cross-react with NF-H antibodies, and moved primarily in the Group IV (SCb) wave of axoplasmic transport. The four NF-H variants were generated by phosphorylation of a single polypeptide. Each of these polypeptides incorporated 32P when retinal ganglion cells were radiolabeled in vivo with [32P]orthophosphate and each cross-reacted with monoclonal antibodies specifically directed against phosphorylated epitopes on NF-H. When dephosphorylated in vitro with alkaline phosphatase, the four variants disappeared, giving rise to a single polypeptide with the same apparent molecular mass (160 kD) as newly synthesized, unmodified NF-H. The NF-H variants distributed differently along optic axons. P197 predominated at proximal axonal levels; P200 displayed a relatively uniform distribution; and P205 and P210 became increasingly prominent at more distal axonal levels, paralleling the distribution of the stationary neurofilament network.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Multiple phosphorylated variants of the high molecular mass subunit of neurofilaments in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments. 314 56

A preliminary study on 9 suckling Wistar rats, which received E. coli stable toxin, and on 12 sham-operated controls showed that acid phosphatase, the marker enzyme for lysosome, was significantly increased in the infected group whereas alkaline phosphatase, glucose 6-phosphatase, succinic dehydrogenase, and proteinase, the marker enzymes for brush border, microsome, mitochondria, and the soluble fraction, respectively, remained unaffected. The results suggest that lysosome, the subcellular organelle responsible for intracellular digestion could be modified by E. coli stable toxin. In another set of experiments, where 7 infected suckling rats and 7 sham-operated controls were used, the maximal activities of lysosomal enzymes (released by Triton X-100) were found to be increased in the infected group confirming the results obtained in the preliminary experiment. The values of the ratio between maximal and basal activity (an expression of the degree of retention of enzymes to lysosome) of acid phosphatase and cathepsin D were also significantly increased, indicating that lysosomal membrane may also be stabilized during the infection. The increased activities of lysosomal enzymes and the increased lysosomal membrane stability suggest that intracellular digestion by lysosome could be increased during E. coli stable toxin infection.
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PMID:Effect of enterotoxigenic Escherichia coli heat stable toxin on intestinal lysosomal enzymes in the suckling rat. 328 51

Isoenzymes of alkaline phosphatase (EC 3.1.3.1) were separated by micro-scale two-dimensional electrophoresis, with isoelectric focusing in capillary gels in the first dimension and polyacrylamide gradient-gel electrophoresis in the second. The isoenzymes detected were identified by several treatments--e.g., incubation with sialidase, papain, Triton X-100, and wheat-germ agglutinin--and by comparison with alkaline phosphatase from liver microsomes. Liver and bone isoforms in normal sera showed overlapping isoelectric points but differed in molecular mass, estimated as 172 and 185 kDa, respectively. Sera of patients with liver disease showed several additional groups of alkaline phosphatase isoforms, two of which were found to consist of multi-molecular complexes. Others probably correspond to incompletely glycated enzyme proteins. A further isoform with a mass of about 250 kDa does not seem to correspond to any known isoform of alkaline phosphatase in serum. With this technique, we demonstrated intra- and interindividual variations of the placental alkaline phosphatase isoenzyme in pregnancy sera.
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PMID:Micro-scale two-dimensional electrophoresis of alkaline phosphatase from serum. 335 9

Coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34 depends on interaction of a lectin on A. viscosus T14V with a cell surface carbohydrate on S. sanguis 34. This carbohydrate was isolated, and its chemical makeup was established. The carbohydrate remained attached to S. sanguis 34 cells through extraction with Triton X-100 and treatment with pronase. It was cleaved from the cell residue by autoclaving and purified by differential centrifugation and column chromatography on DEAE-Sephacel and Sephadex G-75. The polysaccharide contained phosphate which was neither inorganic nor monoester. Treatment with NaOH-NaBH4, followed by Escherichia coli alkaline phosphatase, or with 48% HF at 4 degrees C, followed by NaBH4, yielded inorganic phosphate and oligosaccharide alditols. Therefore, the polysaccharide is composed of oligosaccharide units joined together by phosphodiester bridges. The structure and stereochemistry of the main oligosaccharide alditol was established previously (F. C. McIntire, C. A. Bush, S.-S. Wu, S.-C. Li, Y.-T. Li, M. McNeil, S. Tjoa, and P. V. Fennessey, Carbohydr. Res. 166:133-143). Permethylation analysis, 1H and 31P nuclear magnetic resonance studies on the whole polysaccharide revealed the position of the phosphodiester linkages. The polysaccharide is mainly a polymer of (6) GalNAc(alpha 1-3)Rha(beta 1-4)Glc(beta 1-6)Galf(beta 1-6)GalNAc(beta 1- 3)Gal(alpha 1)-OPO3. It reacted as a single antigen with antiserum to S. sanguis 34 cells and was a potent inhibitor of coaggregation between A. viscosus T14V and S. sanguis 34. Quantitative inhibition of precipitation assays with oligosaccharides, O-allyl N-acetylgalactosaminides, and simple sugars indicated that specific antibodies were directed to the GalNAc end of the hexasaccharide unit. In contrast, coaggregation was inhibited much more effectively by saccharides containing betaGalNAc. Thus, the specificity of the A. viscosus T14V lectin is strikingly different from that of antibodies directed against the S. sanguis 34 polysaccharide.
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PMID:A polysaccharide from Streptococcus sanguis 34 that inhibits coaggregation of S. sanguis 34 with Actinomyces viscosus T14V. 336 Jul 42

Murine T-lymphomas and Thy-1- mutants were labeled overnight with [3H]ethanolamine to detect proteins which possess a glycophospholipid anchor. When labeled cells were treated with 10% trichloroacetic acid and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, both Thy-1 and a second intensely labeled protein (46 kDa) were observed. The presence of the radiolabeled 46-kDa protein in wild type and class E Thy-1 negative cells (cells in which Thy-1 is synthesized but cannot be labeled with [3H]ethanolamine) suggested incorporation into a distinct moiety. Labeling of the 46-kDa protein with [3H]ethanolamine is rapidly inhibited by cycloheximide. Further characterization of the 46-kDa protein by subcellular fractionation and Triton X-114 partitioning indicated that the protein is located in the cytosol. The protein is basic and does not bind to either concanavalin A or wheat germ agglutinin. Labeling of a 46-kDa protein has also been demonstrated in Chinese hamster ovary, COS, rat myeloma, cloned human T-lymphocytes, and HeLa cells. Pronase digestion of the [3H]ethanolamine-labeled 46-kDa protein of wild type lymphoma cells generated a nonbasic and polar labeled fragment which is labile to strong acid and base ([3H]ethanolamine is liberated), insensitive to periodate oxidation and alkaline phosphatase, and does not bind to concanavalin A or wheat germ agglutinin. Judging from methylation studies, the labeled ethanolamine residue does not contain a free amino group. Based on these results, we report a novel post-translational modification of selected protein(s) by the covalent addition of [3H]ethanolamine.
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PMID:Extensive labeling with [3H]ethanolamine of a hydrophilic protein of animal cells. 337 24

The effects of fluoride (20 mumol/L) and bovine bone extract (17 micrograms/ml) were determined on cultures of human bone cells, embryonic chick bone cells, and human skin fibroblasts. The incorporation of [3H]thymidine into DNA was measured 16 hours after the addition of factors. After three to five days treatment, Triton X-100 extracts of the cells were assayed for acid phosphatase activity, in the presence and absence of tartrate, and for alkaline phosphatase activity. Fluoride stimulated [3H]thymidine incorporation and specific activity of alkaline phosphatase in human bone cells and chick bone cells but not in human skin cells. Fluoride also stimulated the cell population doubling rate of the human bone cells with an optimum of approximately 20 mumol/L. Bovine bone extract stimulated thymidine uptake into DNA several-fold and decreased alkaline phosphatase activity in all three types of cultured cells. The specific activity of tartrate-resistant acid phosphatase was increased in bone cells but not in skin fibroblasts. These results suggest that fluoride specifically stimulates the proliferation and differentiation of osteoblasts, while the growth factors in bovine bone extract primarily stimulate proliferation of bone cells. Cultures of human bone cells respond similarly to chick calvarial cells when treated with fluoride or bovine bone extract.
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PMID:Fluoride and bovine bone extract influence cell proliferation and phosphatase activities in human bone cell cultures. 340 30

The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzes p-nitrophenyl phosphate over a broad pH range. Acid phosphatase activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2-mercaptoethanol, and ethylenediaminetetra-acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X-100, while such treatment causes a significant loss of acid phosphatase activity. Both activities are nonspecific and hydrolyze a variety of phosphorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.
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PMID:Acid phosphatase activity in the isolated brush border membrane of the tapeworm, Hymenolepis diminuta: partial characterization and differentiation from the alkaline phosphatase activity. 341 89


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