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)

Protein A and C, which are major components of the acidic proline-rich proteins in human saliva, were digested, before or after adsorption to hydroxyapatite, with alkaline phosphatase, trypsin, thermolysin and a proteinase preparation from salivary sediment. The results demonstrate that the binding site is located in the proline-poor N-terminal part of the protein, possibly between residues 3 and 25. Phosphoserine is necessary for maximal adsorption of the proteins to hydroxyapatite. When proteins A and C are adsorbed to hydroxyapatite before proteolytic digestion there is a protection of some of the susceptible bonds in the N-terminal part of the proteins and a gradual removal of the proline-rich C-terminal part. Thermolysin can cleave susceptible bonds in the part of the protein that remains bound to hydroxyapatite, but at least some of the resulting peptides are retained on the mineral. Since the ability of the proteins to inhibit hydroxyapatite formation and to bind calcium is located in the N-terminal proline-poor part, it is possible that these activities are retained after proteolytic digestion of the adsorbed proteins.
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PMID:The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins. 23 Aug 18

Absence of precipitation of calcium phosphate salts onto tooth surfaces from human saliva, which is supersaturated with respect to calcium phosphate salts, has been attributed in part to the presence in the salivary secretions of a group of acidic proline-rich phosphoproteins (PRP). These macromolecules are considered to act by adsorbing onto dental enamel where they inhibit surface-induced precipitation of calcium phosphate salts. The inhibitory activity is known to be associated primarily with the amino-terminal region of the PRP. The aim of this study was to determine the features of the primary structure of this molecular segment responsible for inhibitory activity. The 30-residue, amino-terminal segment of PRP-3, which contains the two phosphoserines and 11 of the 13 carboxyl groups present in PRP-3, was obtained by tryptic digestion. This peptide, designated PRP-3(TI), was treated with thermolysin to give the monophosphopeptides, Val-PSer-Gln-Glu-Asp-Val-Pro and Leu-Val-Ile-Ser-Asp-Gly-Gly-Asp-PSer-Glu-Gln, and with alkaline phosphatase to give the dephosphorylated analog, PRP-3(TI)DP. The inhibitory activities of PRP-3(TI) and the derived peptides, a synthetic peptide, phosphoseryl-phosphoserine (PSer-PSer), and O-phosphoserine (PSer), were determined using an assay based on inhibition of seeded precipitation of calcium phosphate. Inhibitory activities, expressed as concentrations of inhibitors required to give standard inhibitory activities, were PRP-3(TI), 0.59 microM; PSer-PSer, 3.5 microM; the two monophosphopeptides, 29 and 32.5 microM; PRP-3(TI)DP, 56 microM; PSer, 329 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of calcium phosphate precipitation by human salivary acidic proline-rich proteins: structure-activity relationships. 310 42

Microtubule-associated protein 2 (MAP2) is an in vitro substrate for MAP kinase. Part of the phosphorylation occurs at the C-terminal microtubule-binding domain of the molecule which contains a cluster of putative consensus sites for MAP kinase on a proline-rich region. A peptide with the sequence RTPGTPG-TPSY, located at this region of the molecule, is efficiently phosphorylated by MAP kinase in vitro. An antibody (972) raised against this non-phosphorylated peptide has been used to test for in vivo phosphorylation at the proline-rich domain of the MAP2 molecule. The reaction of purified MAP2 with antibody 972 diminishes after in vitro phosphorylation by MAP kinase and is enhanced after in vitro dephosphorylation by alkaline phosphatase. A fraction of brain MAP2 isolated by iron-chelation affinity chromatography appears to be phosphorylated in vivo at the site recognized by antibody 972. There is some variation in the phosphorylation of MAP2 at the proline-rich region throughout rat brain development. MAP2C is more highly phosphorylated in the developing rat brain, whereas high-molecular-mass MAP2 is more extensively phosphorylated in the adult rat brain.
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PMID:Variations in in vivo phosphorylation at the proline-rich domain of the microtubule-associated protein 2 (MAP2) during rat brain development. 788 2

A 190-kDa surface protein antigen (PAc) of Streptococcus mutans binds to human salivary components. For detection of specific binding of the PAc protein to human salivary components, a simple sandwich assay was used. Microtiter plates precoated with recombinant PAc (rPAc), PAc fragments, or S. mutans whole cells were allowed to react with human whole saliva and then were incubated with biotinylated rPAc. The biotinylated rPAc bound to salivary components was detected by use of alkaline phosphatase-conjugated streptavidin and p-nitrophenylphosphate. In this assay, the binding of whole cells of S. mutans and purified rPAc to salivary components was confirmed. For determination of a saliva-binding region of the PAc molecule, 14 truncated PAc fragments were constructed by use of the polymerase chain reaction and an expression vector, pAX4a+. The binding of these truncated PAc fragments to human salivary components was determined by the sandwich assay. Among the truncated PAc fragments, fragments corresponding to residues 39 to 864 and residues 39 to 1000 of PAc showed a high ability to bind to salivary components. Shorter recombinant fragments corresponding to residues 39 to 217, residues 200 to 481, residues 470 to 749, and residues 688 to 864 did not exhibit any binding ability. The fragment that corresponds to a proline-rich repeating region (residues 828 to 1000) bound directly to the PAc protein. These results suggest that residues 39 864 of the PAc molecule are important in the binding of the surface protein to human salivary components, and the proline-rich repeating region of the PAc protein may contribute to spontaneous self-aggregation of the PAc protein.
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PMID:Saliva-binding region of Streptococcus mutans surface protein antigen. 840 23

Sixteen-amino-acid-long peptides, corresponding to the optimal ligand preferences of the Src homology 3 (SH3) domains of Abl, Cortactin, Crk, p53BP2, and Src, were fused to the N-terminus of Escherichia coli alkaline phosphatase (AP). These secreted fusion proteins have been used as one-step detection probes of peptide ligand-SH3 domain interactions on microtiter plates and membranes. The binding of both the class I and II SH3 ligand-AP fusion proteins to their targets is robust and specific in comparison to chemically synthesized biotinylated peptides, used either in monovalent or tetravalent formats. p53BP2 and Cortactin SH3 ligand-AP fusions have been used to screen a mouse embryo lambda cDNA expression library and resulted in the cloning of p53BP2 and several known proteins with SH3 domains similar to that of Cortactin, respectively. In addition, the approximately 60-amino-acid-long SH3 domains of Src and Abl were fused to AP and the resulting fusion proteins were found to bind specifically to their respective peptide ligands in microtiter plates and proteins containing proline-rich regions in screens of a lambda cDNA expression library. Thus, SH3 peptide ligand- and SH3 domain-AP fusion proteins are convenient and sensitive reagents for examining the specificity of SH3 domain-ligand interactions, identifying potentially interacting proteins, and establishing high-throughput screens of combinatorial chemical libraries.
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PMID:Examining the specificity of Src homology 3 domain--ligand interactions with alkaline phosphatase fusion proteins. 912 84

The major type of acetylcholinesterase in vertebrates (AChET) is characterized by the presence of a short C-terminal domain of 40 residues, the 'tryptophan amphiphilic tetramerization' (WAT) domain. The presence of this domain is not necessary for catalytic activity but is responsible for hydrophobic interactions and for the capacity of AChET subunits to form quaternary associations with anchoring proteins, thereby conditioning their functional localization. In the collagen tail of asymmetric forms, we characterized a small conserved region that is sufficient for binding an AChET tetramer, the proline-rich attachment domain (PRAD). We show that the WAT domain alone is sufficient for association with the PRAD, and that it can attach foreign proteins (alkaline phosphatase, GFP) to a PRAD-containing construct with a glycophosphatidylinositol anchor (GPI), and thus anchor them to the cell surface. Furthermore, we show that isolated WAT domains, or proteins containing a WAT domain, can replace individual AChET subunits in PRAD-linked tetramers. This suggests that the four WAT domains interact with the PRAD in a similar manner. These quaternary interactions can form without intercatenary disulfide bonds. The common catalytic domains of AChE are not necessary for tetrameric assembly, although they may contribute to the stability of the tetramer.
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PMID:A four-to-one association between peptide motifs: four C-terminal domains from cholinesterase assemble with one proline-rich attachment domain (PRAD) in the secretory pathway. 979 27

Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.
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PMID:The secretory carrier membrane protein family: structure and membrane topology. 1098 91

Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelical lid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.
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PMID:The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. 1125 15

A pine extensin-like protein (PELP) has been localized in metabolically active cells of differentiating xylem and in mature wood of loblolly pine (Pinus taeda L.). This proline-rich glycosylated protein was purified from cell walls of differentiating xylem by differential solubility and gel electrophoresis. Polyclonal rabbit antibodies were raised against the deglycosylated purified protein (dPELP) and purified antibody was used for immunolocalization. Immunogold and alkaline phosphatase secondary antibody staining both show antigen in secondary cell walls of earlywood and less staining in latewood. Immunoassays of milled dry wood were developed and used to show increased availability of antigen after hydrogen fluoride or cellulase treatment and decreased antigen after chlorite treatment. The specificity of the antigen-antibody reaction was confirmed by competition assays and by preadsorption of antibody to the purified protein. We propose that extensin-like protein is present in xylem cell walls during lignification and that the protein remains as a structural component of cell walls in wood for many years after xylogenesis. We suggest that such structural proteins play important roles in the differentiation of xylem and thereby could affect the properties of wood.
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PMID:Wood contains a cell-wall structural protein. 1160 6

Lymphocyte stimulation by immunoreceptors is achieved through the activation of multiple signaling pathways leading to cytokine gene transcription. Adapter proteins are critical signaling components that can integrate multiple pathways by allowing the assembly of multimolecular signaling complexes. We previously showed that the cytoplasmic adapter 3BP2 (also known as SH3BP2) promotes NFAT/AP-1 transcriptional activities in T cells through the activation of Ras- and calcineurin-dependent pathways. However, the molecular mechanisms by which 3BP2/SH3BP2 regulates cell signaling and activation remain poorly documented. In this study, using a combination of yeast two-hybrid analysis and biochemical approaches, we present evidence for a physical interaction between 3BP2 and the chaperone protein 14-3-3. This interaction was direct and constitutively detected in yeast and in mammalian cells. Phorbol ester, pervanadate, and forskolin/isobutylmethylxanthine stimulations enhanced this interaction, as well as co-expression of constitutive active mutants of serine/threonine kinases, including protein kinase C. We found that dephosphorylation of 3BP2 by alkaline phosphatase disrupted its interaction with 14-3-3 and that 3BP2 was a substrate of purified protein kinase C in vitro, suggesting that the phosphorylation of 3BP2 by upstream kinases was required for 14-3-3 binding. Using deletion mutants of 3BP2, two 14-3-3 binding domains were mapped to two proline-rich (residues 201-240 and 270-310) domains of 3BP2. These domains were shown to contain two 14-3-3 consensus binding motifs. We identified residues Ser(225) and Ser(277) of 3BP2 as being essential for interaction with 14-3-3 family proteins, optimal 3BP2 serine phosphorylation, and then for 3BP2-dependent function. Indeed, a 3BP2 mutant protein incapable of binding 14-3-3 showed increased capacity to stimulate NFAT transcriptional activities, suggesting that 14-3-3 binding to 3BP2 negatively regulates 3BP2 adapter function in lymphocytes.
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PMID:The chaperone protein 14-3-3 interacts with 3BP2/SH3BP2 and regulates its adapter function. 1250 Dec 43


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