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)

To identify elements participating in the process of transformation, a bank of genetically altered mutants of Streptococcus pneumoniae with defects in exported proteins was assessed for a decrease in transformation efficiency. One mutant consistently transformed 10-fold less than the parent strain. Sequence analysis and reconstitution of the altered locus revealed a gene, plpA (permease-like protein), which encodes a putative substrate-binding protein belonging to the family of bacterial permeases responsible for peptide transport. The derived amino acid sequence for this gene was 80% similar to AmiA, a peptide-binding protein homologue from pneumococcus, and 50% similar over 230 amino acids to Spo0KA which is a regulatory element in the process of transformation and sporulation in Bacillus subtilis. PlpA fusions to alkaline phosphatase (PhoA) were shown to be membrane associated and labelled with [3H]-palmitic acid, which probably serves as a membrane anchor. Experiments designed to define the roles of the plpA and ami determinants in the process of transformation showed that: (i) mutants with defects in plpA were > 90% transformation deficient while ami mutants exhibited up to a fourfold increase in transformation efficiency; (ii) compared to the parental strain, the onset of competence in an ami mutant occurred earlier in logarithmic growth, whereas the onset was delayed in a plpA mutant; and (iii) the plpA mutation decreases the expression of a competence-regulated locus. Since the permease mutants would fail to bind specific ligands, it seems likely that the substrate-permease interaction modulates the process of transformation.
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PMID:Peptide permeases modulate transformation in Streptococcus pneumoniae. 752 29

Glycosylphosphatidylinositol (GPI)-anchored proteins occur widely, perhaps universally, on the surface of animal cells, where they perform a variety of important functions. However, the existence of GPI-anchored proteins on plant cells has never been established. Evidence is presented in this communication for the occurrence of a 50 kDa GPI-anchored alkaline phosphatase (AP) induced in the duckweed Spirodela oligorrhiza by phosphate deprivation. Triton X-114 partitioning of the Spirodela proteins yielded two forms of AP activity. The detergent-associated form was labeled prominently by [3H]ethanolamine, [3H]myristic acid and [3H]palmitic acid. This amphiphilic form of AP, like authentic GPI-anchored AP from mammals, was clearly resolved from the remaining, water-soluble AP activity by two types of incompletely-denaturing polyacrylamide gel electrophoresis. Lipid covalently bound to the solvent-delipidated amphiphilic AP was resistant to cleavage by phosphatidylinositol-specific phospholipase C. Strong acid or alkaline hydrolysis of the 3H-fatty acid-labeled amphiphilic AP yielded radioactive fatty acids and a radioactive lipid tentatively identified as a long chain base. The more abundant water-soluble AP was also radioactive in plants incubated with [3H]ethanolamine and was labeled to a lesser extent by 3H-fatty acids. The water-soluble AP, unlike its amphiphilic counterpart, could be freed of all fatty acid radioactivity by mild alkaline hydrolysis, indicating the continued presence of an ester-linked fatty acid. All evidence supports the conclusion that Spirodela AP is synthesized as an amphiphilic protein with a ceramide-containing GPI anchor.
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PMID:Evidence for a glycosylinositolphospholipid-anchored alkaline phosphatase in the aquatic plant Spirodela oligorrhiza. 864 7

The Agrobacterium tumefaciens virB7 gene product contains a typical signal sequence ending with a consensus signal peptidase II cleavage site characteristic of bacterial lipoproteins. VirB7 was shown to be processed as a lipoprotein by (i) in vivo labeling of native VirB7 and a VirB7::PhoA fusion with [3H]palmitic acid and (ii) inhibition of VirB7 processing by globomycin, a known inhibitor of signal peptidase II. A VirB7 derivative sustaining a Ser substitution for the invariant Cys-15 residue within the signal peptidase II cleavage site could not be visualized immunologically and failed to complement a delta virB7 mutation, establishing the importance of this putative lipid attachment site for VirB7 maturation and function. VirB7 partitioned predominantly with outer membrane fractions from wild-type A348 cells as well as a delta virB operon derivative transformed with a virB7 expression plasmid. Expression of virB7 fused to phoA, the alkaline phosphatase gene of Escherichia coli, gave rise to high alkaline phosphatase activities in E. coli and A. tumefaciens cells, providing genetic evidence for the export of VirB7 in these hosts. VirB7 was shown to be intrinsically resistant to proteinase K; by contrast, a VirB7::PhoA derivative was degraded by proteinase K treatment of A. tumefaciens spheroplasts and remained intact upon treatment of whole cells. Together, the results of these studies favor a model in which VirB7 is topologically configured as a monotopic protein with its amino terminus anchored predominantly to the outer membrane and with its hydrophilic carboxyl domain located in the periplasmic space. Parallel studies of VirB5, VirB8, VirB9, and VirB10 established that each of these membrane-associated proteins also contains a large periplasmic domain whereas VirB11 resides predominantly or exclusively within the interior of the cell.
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PMID:The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface. 865 94

A novel peptidoglycan-linked lipoprotein (ComL) has been identified which is required for efficient transformation of Neisseria gonorrhoeae by species-related DNA. Although most mutations in comL appear to be lethal, transposon shuttle mutagenesis was successful in generating a single viable comL mutant of N. gonorrhoeae strain MS11. This mutant, N457, exhibits a cratered and crinkled colony morphology and grows slower than wild-type MS11. However, as indicated by electron microscopy, this retardation is due to a small bacterial size rather than to a decreased generation time of the mutant bacteria. Complementation of N457 with an intact comL gene via the Hermes shuttle system fully reconstitutes bacterial size, colony morphology, and transformation competence of the wild-type strain. comL is a single-copy gene and maps downstream of the previously described comA gene. It is transcribed in the opposite direction, probably using the same transcriptional terminator. ComL has a predicted size of 29 kDa and is synthesized in Escherichia coli under the control of its native promoter, which is highly conserved with the E. coli promoter consensus sequence. The 5' end of the coding sequence reveals a lipoprotein secretion signal shown to be functional by gene fusion with alkaline phosphatase (phoA'). In E. coli, cloned ComL can be labelled with [3H]-palmitic acid, thus demonstrating its lipoproteinaceous nature. Palmitoylated ComL appears to be covalently bound to the murein sacculus of E. coli and N. gonorrhoeae since it resists boiling in 4% sodium dodecyl sulphate and is released only by lysozyme treatment. Homologous counterparts of the comL gene are found in Neisseria meningitidis as well as in several nonpathogenic Neisseria species.
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PMID:A novel peptidoglycan-linked lipoprotein (ComL) that functions in natural transformation competence of Neisseria gonorrhoeae. 883 Feb 66

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24-197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the alpha-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.
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PMID:Identification of a palmitic acid-modified form of human Sonic hedgehog. 959 55

Age-related bone loss eventually leads to osteopenia in men and women. The etiology of age-related bone loss is currently unknown; however, decreased osteoblast activity contributes to this phenomenon. In turn, osteoblast proliferation and function is dependent on energy production, thus the loss of energy production that occurs with age may account for the deficient osteoblast activity. Carnitine and dehydroepiandrosterone-sulfate (DHEAS), both of which decline with age, promote energy production through fatty acid metabolism. Thus, we hypothesized that carnitine and DHEAS would increase osteoblast activity in vitro. Accordingly, we measured the effect of carnitine and DHEAS on palmitic acid oxidation as a measure of energy production, and alkaline phosphatase (ALP) activity and collagen type I (COL) as indices of osteoblast function in primary porcine osteoblast-like cell cultures. Carnitine (10(-3) and 10(-1) M) but not DHEAS (10(-9), 10(-8), and 10(-7) M) increased carnitine levels within the cells. Carnitine alone and in combination with DHEAS increased palmitic acid oxidation. Both carnitine and DHEAS alone and in an additive fashion increased ALP activity and COL levels. These results demonstrate that in osteoblast-like cells in vitro, energy production can be increased by carnitine and osteoblast protein production can be increased by both carnitine and DHEAS. These data suggest that carnitine and DHEAS supplementation in the elderly may stimulate osteoblast activity and decrease age-related bone loss.
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PMID:Carnitine and dehydroepiandrosterone sulfate induce protein synthesis in porcine primary osteoblast-like cells. 1034 Oct 26

Prior studies have shown that 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] plays a major role in resting zone chondrocyte differentiation and that this vitamin D metabolite regulates both phospholipase A2 and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipase A2 action and has been shown in other systems to affect a variety of cellular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH)2D3 on markers of proliferation, differentiation, and matrix production in resting zone chondrocytes and to characterize the mechanisms by which arachidonic acid regulates PKC, which was shown previously to mediate the rapid effects of 24,25-(OH)2D3 and arachidonic acid on these cells. Confluent, fourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [3H]thymidine incorporation and inhibited the activity of alkaline phosphatase and PKC, but had no effect on proteoglycan sulfation. In contrast, 24,25-(OH)2D3 inhibited [3H]thymidine incorporation and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC activity. In cultures treated with both agents, the effects of 24,25-(OH)2D3 were reversed by arachidonic acid. The PKC isoform affected by arachidonic acid was PKCalpha; cytosolic levels were decreased, but membrane levels were unaffected, indicating that translocation did not occur. Arachidonic acid had a direct effect on PKC in isolated plasma membranes and matrix vesicles, indicating a nongenomic mechanism. Plasma membrane PKCalpha was inhibited, and matrix vesicle PKCzeta was stimulated; these effects were blocked by 24,25-(OH)2D3. Studies using cyclooxygenase and lipoxygenase inhibitors indicate that the effects of arachidonic acid are due in part to PG production, but not to leukotriene production. This is supported by the fact that H8-dependent inhibition of protein kinase A, which mediates the effects of PGE2, had no effect on the direct action of arachidonic acid but did mediate the role of arachidonic acid in the cell response to 24,25-(OH)2D3. Diacylglycerol does not appear to be involved, indicating that phospholipase C and/or D do not play a role. Gamma-linolenic acid, an unsaturated precursor of arachidonic acid, elicited a similar response in matrix vesicles but not plasma membranes, whereas palmitic acid, a saturated fatty acid, had no effect. These data suggest that arachidonic acid may act as a negative regulator of 24,25-(OH)2D3 action in resting zone chondrocytes.
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PMID:Arachidonic acid directly mediates the rapid effects of 24,25-dihydroxyvitamin D3 via protein kinase C and indirectly through prostaglandin production in resting zone chondrocytes. 1038 91

Surfactant-like particle (SLP) is a phosphatidylcholine (PC)-rich membrane produced in the small intestine, and its secretion is increased by fat feeding. In Caco-2 cells known to produce SLP, preincubation with [(3)H]palmitate labelled the SLP and was used as a marker for newly secreted membrane. SLP-associated PC and protein (d=1.07-1.08 g/ml in a linear non-equilibrium NaBr gradient) were secreted in parallel with triacylglycerols (TG) and at a rate about twice the control rate in response to feeding cells with an oleate/egg PC mixture. Cholesterol and apolipoprotein A-I identified only a small peak corresponding to high-density lipoprotein (HDL), but the largest peak corresponded with SLP (d=1.07-1.08). Palmitate incorporation into PC showed a similar small peak migrating at the density of HDL, but most labelled PC secreted from the cells was due to SLP. PC secretion, alkaline phosphatase activity, and newly synthesized immunoprecipitated SLP proteins from conditioned serum-free media migrated together at a density of >/=1.21 g/ml in a lipoprotein NaBr step gradient, and represented SLP. Glycerol incorporated into TG migrated at a peak density of 1.12 g/ml, consistent with HDL secretion from cells incubated in serum-free media. These data confirm that the secreted PC in SLP is distinct from lipoprotein particles. Incorporation of [(3)H]palmitate into the PC fraction of either whole cell homogenate or isolated brush border membranes was not affected by oleate/egg PC feeding. Both Pluronic L-81, an inhibitor of chylomicron secretion, and BMS-197636-02, a microsomal triglyceride transfer protein inhibitor, blocked the secretion of both TG and PC. Elevation of intracellular cAMP levels that stimulate surfactant secretion from type II pneumocytes caused a 50% reduction in SLP and TG secretion from Caco-2 cells. These results confirm the SLP response to fat feeding found in vivo, further supporting a role for SLP in TG secretion from the enterocyte, and show that the regulation of SLP secretion differs from that of pulmonary surfactant.
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PMID:Regulation of surfactant-like particle secretion by Caco-2 cells. 1128 80

Biochemical assessment of liver damage during ethanol-induced stress was done by measuring the activities of serum enzymes, viz., aspartate transaminase (AST) and alkaline phosphatase (ALP), which were significantly elevated in rats fed ethanol. Ethanol administration for a period of 60 days modifies the fatty acid composition, and the analysis of fatty acids showed that there was a significant increase in the concentrations of palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1) in liver, kidney, and brain, whereas the concentrations of palmitoleic (16:1) and arachidonic acid (20:4) were significantly decreased. The breakdown products of arachidonic acids (20:4), prostaglandins, were elevated. The antioxidants curcumin and N-acetylcysteine (NAC) decreased the activities of serum AST and ALP. Curcumin and NAC decreased the concentrations of fatty acids, viz., palmitic, stearic, and oleic acid, whereas arachidonic acid and palmitoleic acid were elevated. The prostaglandin concentrations were also decreased after curcumin and N-acetylcysteine treatment. Thus the present investigation shows that curcumin and N-acetylcysteine prevent the fatty acid changes produced by ethanol and also reduce the inflammatory response of ethanol by reducing the level of prostaglandins.
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PMID:Potential role of antioxidants during ethanol-induced changes in the fatty acid composition and arachidonic acid metabolites in male Wistar rats. 1150 46

Detergent-resistant membranes (DRM) were prepared from bovine kidney cortex. The criterion used to test their purification was the increase in the activity of a GPI membrane-anchored protein, the alkaline phosphatase. Its association with specific proteins and lipids was tested. Two successive Triton X-100 treatments followed by purification on sucrose gradient at 4 degrees C were necessary to obtain DRM with a maximum of alkaline phosphatase activity and a typical protein pattern. A third Triton treatment did not alter this DRM composition. Among the enriched protein, we identified, by mass spectrometry, a microsomal dipeptidase, which was GPI membrane-anchored. Protein-kinase activities, mainly serine-kinase, were enriched during the DRM purification. Using the typical FTIR olefinic =C-H bands of the acyl chains, a global decrease in the unsaturation level of DRM lipids was observed as compared with total membranes. Three main phospholipids were identified in DRM. Their fatty acid compositions were determined by gas chromatography and compared with those of total membranes. The most enriched saturated fatty acid was palmitic acid (+44% for phosphatidylethanolamine, +52% for phosphatidylcholine and +49% for sphingomyelin), agreeing with a selection of specific phospholipids among the saturated ones during the DRM purification.
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PMID:Protein and lipid analysis of detergent-resistant membranes isolated from bovine kidney. 1473 76


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