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)

The 26S proteasome complex, consisting of two multisubunit complexes, a 20S proteasome and a pair of 19S regulatory particles, plays a major role in the nonlysosomal degradation of intracellular proteins. The 20S proteasome was purified from yeast and separated by two-dimensional gel electrophoresis (2-DE). A total of 18 spots separated by 2-DE were identified as the 20S proteasome subunits by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The alpha2-, alpha4- and alpha7-subunits gave multiple spots, which converged into one spot for each subunit when treated with alkaline phosphatase. The difference of pI between phosphorylated and dephosphorylated spots and their reaction against anti-phosphotyrosine antibody suggested that the alpha2- and alpha4-subunits are phosphorylated either at Ser or at Thr residue, and the alpha7-subunit is phosphorylated at Tyr residue(s). These phosphorylated subunits were analyzed by electrospray ionization-quadrupole time of flight-tandem MS (ESI-QTOF-MS/MS) to deduce the phosphorylation sites. The 20S proteasome has three different protease activities: chymotrypsin-like, trypsin-like and peptidylglutamyl peptide-hydrolyzing activities. The phosphatase treatment increased K(m) value for chymotrypsin-like activity of the 20S proteasome, indicating that phosphorylation may play an important role in regulating the proteasome activity.
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PMID:Electrophoretic analysis of phosphorylation of the yeast 20S proteasome. 1184 May 41

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Furthermore, numerous results demonstrate that lipid peroxidation products affect central biochemical pathways and intracellular signalling at physiological concentrations. 4-Hydroxynonenal (HNE) is one of the most active products of lipid peroxidation. This work has focused on the evaluation of HNE nuclear content, so far never directly measured, by electrospray-ionization-mass-spectrometry (ESI/MS) and on the correlation between its concentration and the induced effects after exogenous administration. In a human osteosarcoma cell line (SaOS2), HNE exhibited an early cytotoxic effect characterized by apoptosis, cytostatic and differentiating effects characterized by slow growth, increase in alkaline phosphatase (ALP), and alpha5 integrin subunit content with decrease in tumorigenicity.
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PMID:Cytotoxic and cytostatic effects induced by 4-hydroxynonenal in human osteosarcoma cells. 1205 86

Protein phosphorylation is the most important reversible post-translational modification in cells. Analysis of phosphorylated proteins and identification of their phosphorylation sites is helpful for understanding their biological functions. MALDI-TOF-MS and ESI-Q-TOF-MS play important roles in protein phosphorylation analysis. In this work, immobilized metal affinity chromatography (IMAC) was used to selectively enrich phosphopeptides from protein digest mixtures, and treatment of phosphopeptides with alkaline phosphatase was used to confirm the phosphorylation. Finally, the phosphorylation sites were determined by tandem mass spectrometry analysis and database searching.
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PMID:[Analysis of protein phosphorylation by combination of IMAC, phosphatase with biological mass spectrometry]. 1276 8

DNA-phosphate adducts are known to be formed by a variety of alkylating agents. Due to little or no repair of DNA-phosphate adducts, these adducts may offer increased possibilities of both identifying and quantifying DNA adducts. The formation of DNA-phosphate adducts leads to a complete esterification of the phosphate group giving rise to a phosphotriester configuration. This work consists of the characterization of ethyl phosphotriesters (Ethyl PTE) using miniaturized LC-ESI-MS/MS and column switching in enzymatic hydrolysate of DNA treated in vitro with the model compound N-ethyl-N-nitrosourea (ENU). In vitro ENU-treated DNA was enzymatically degraded using nuclease P1, phosphodiesterase, and alkaline phosphatase. The use of column switch allowed for large-volume injections, where unmodified nucleosides were discarded in the loading step. The analytes were forward flushed to the analytical column in the eluting step and separated using a linear gradient. Ten different ethyl PTEs (dGpEtdG, dApEtdA, dCpEtdC, TpEtT, dGpEtdA, dGpEtdC, dGpEtT, dApEtdC, dApEtT, and dCpEtT) were characterized by their masses and CAD product ion spectra. Measurements of accurate masses were carried out yielding experimental masses within 5 ppm of the calculated masses for 9 of the 10 ethyl PTEs. For comparison, the enzymatic hydrolysate of ENU-treated DNA was subjected to transalkylation of the DNA-phosphate adducts by cob(I)alamin. Formed ethyl-cobalamins were analyzed according to earlier developed methods. The limit of detection of an alkyl-cobalamin standard and an alkyl PTE standard was 2 fmol and 5 fmol, respectively.
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PMID:Analysis of DNA-phosphate adducts in vitro using miniaturized LC-ESI-MS/MS and column switching: phosphotriesters and alkyl cobalamins. 1504 64

Separation of an extract of corpora cardiaca from the protea beetle, Trichostetha fascicularis, by single-step RP (reverse-phase)-HPLC and monitoring of tryptophan fluorescence resulted in two distinctive peaks, the material of which mobilized proline and carbohydrates in a bioassay performed using the beetle. Material from one of these peaks was; however, inactive in the classical bioassays of locusts and cockroaches that are used for detecting peptides belonging to the AKH (adipokinetic hormone) family. After enzymatically deblocking the N-terminal pyroglutamic acid (pGlu) residue in the peptide material and sequencing by Edman degradation, a partial sequence was obtained: (pGlu)-Ile-Asn-Met-Thr-Xaa-Gly-Trp. The complete sequence was deduced from ESI-MS(n) (electrospray ionization multi-stage-MS); position six was identified as a phosphothreonine residue and the C-terminus is amidated. The peptide, code-named Trifa-CC, was chemically synthesized and used in confirmatory experiments to show that the primary structure had been correctly assigned. To our knowledge, this is the first report of a phosphorylated invertebrate neuropeptide. Synthetic Trifa-CC co-elutes with the natural peptide, found in the gland of the protea beetle, after RP-HPLC. Moreover, the natural peptide can be dephosphorylated by alkaline phosphatase and the product of that reaction has the same retention time as a synthetic nonphosphorylated octapeptide which has the same sequence as Trifa-CC. Finally, synthetic Trifa-CC has hypertrehalosaemic and hyperprolinaemic biological activity in the protea beetle, but even high concentrations of synthetic Trifa-CC are inactive in locusts and cockroaches. Hence, the correct peptide structure has been assigned. Trifa-CC of the protea beetle is an unusual member of the AKH family that is unique in its post-translational modification. Since it increases the concentration of carbohydrates and proline in the haemolymph when injected into the protea beetle, and since these substrates are also used during flight, we hypothesize that Trifa-CC controls the mobilization of these metabolites in the protea beetle.
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PMID:Unique translational modification of an invertebrate neuropeptide: a phosphorylated member of the adipokinetic hormone peptide family. 1627 Oct 39

Mammalian cytochrome c (Cyt c) has two primary functions: transfer of electrons from the bc1 complex to cytochrome c oxidase (COX) as part of the mitochondrial electron transport chain (ETC), and participation in type II apoptosis. Several studies have indicated that components of the ETC can be phosphorylated, and we have recently shown that the Cyt c electron acceptor COX is phosphorylated on Tyr-304 of subunit I in liver upon activation of the cAMP-dependent pathway, leading to strong enzyme inhibition. However, covalent modification of Cyt c through phosphorylation has not yet been reported. We have isolated Cyt c from cow heart under conditions that preserve the physiological in vivo phosphorylation status. Western analysis with an anti-phosphotyrosine antibody indicated tyrosine phosphorylation. The site of phosphorylation was definitively assigned by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS) to Tyr-97, one of the four tyrosine residues present in Cyt c. The phosphorylated tyrosine is part of a motif that contains five residues identical to the tyrosine phosphorylation site in COX subunit I. Spectral analysis revealed that the characteristic 695 nm absorption band is shifted to 687 nm and reversed after treatment with alkaline phosphatase. This band results from the Met-80-heme iron bond, and its shift might indicate changes in the catalytic heme crevice. In vivo phosphorylated Cyt c shows enhanced sigmoidal kinetics with COX, and half-maximal turnover is observed at a Cyt c substrate concentration of 5.5 microM compared to 2.5 microM for alkaline phosphatase-treated Cyt c. Possible consequences of Tyr-97 phosphorylation with respect to cardiolipin binding and of location of Tyr-97 in close proximity to Lys-7, a crucial residue for interaction with Apaf-1 during apoptosis, are discussed.
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PMID:New prospects for an old enzyme: mammalian cytochrome c is tyrosine-phosphorylated in vivo. 1686 57

Highly phosphorylated casein with a low molecular mass was isolated from Haflinger mare's milk by RP-HPLC. It accounts for 4.0% of the casein content. Its mass was determined by LC-ESI-MS before and after treatment by alkaline phosphatase. The molecular mass found for the apo-form (10,591 +/- 2 Da) is in agreement with its primary structure, which was established by ESI-MS/MS from tryptic peptides. It appeared that this short protein (94 amino acid residues) is an internally truncated form of the full-length equine beta-casein (226 residues). This low-Mr variant of equine beta-casein displays a large deletion (residues 50-181), due to a cryptic splice site usage occurring within exon 7 during the course of primary transcripts processing. The phosphorylation pattern of this equine beta-casein variant was investigated by LC-ESI-MS and 2-DE. Seven phosphorylation forms were identified with one to seven phosphate groups with pIs ranging between 4.67 and 4.01. The major isoforms carry five and six phosphate groups.
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PMID:The primary structure of a low-Mr multiphosphorylated variant of beta-casein in equine milk. 1736 89

Histatin 1 (His-1) derivatives showing serial mass increases of 80.0 +/- 0.1 Da were detected in human saliva by HPLC-ESI-MS. The same derivatives were also found in granules of submandibular glands and secretions of submandibular/sublingual glands, but not in granules and secretions of parotid glands. Only one phosphate group was present in His-1 and its derivatives, since treatment with alkaline phosphatase provided an 80.0 Da mass decrease. His-1 derivatives were almost completely transformed into His-1 by treatment with 1 M HCl at 100 degrees C, suggesting the presence of O-sulfotyrosine, which is more labile than phospho-Tyr to acidic hydrolysis. CE-MS analysis of pronase extensive digestion of derivatives confirmed the presence of sulfotyrosine. Derivatives were digested by trypsin, proteinase K, and protease V-8 and analyzed by different MS strategies. The results allowed locating sulfation on the last four tyrosines (Tyr 27, 30, 34, and 36). This study is the first report of the gland-specific sulfation of a salivary phosphopeptide in vivo.
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PMID:Tyrosine polysulfation of human salivary histatin 1. A post-translational modification specific of the submandibular gland. 1750 97

A novel detection method for the analysis of multi-phosphopeptides using microcolumn high performance liquid chromatography-electrospray ionization tandem mass spectrometry (microHPLC-ESI-MS/MS) was proposed by the dephosphorylation treatment of alkaline phosphatase (AP). After the selective enrichment by a microcolumn packed with TiO2, phosphopeptides from the tryptic digests of beta-casein were dephosphorylated by AP. Through the removal of phosphate groups, the detection of multi-phosphopeptides according to the non-phosphorylated ones was achieved by ESI-MS/MS. By comparing the chromatograms before and after the AP treatment, mono-phosphopeptides were identified based on the relative molecular mass (Mr) difference of 80. Furthermore, since more peaks appeared after the treatment, the existence of multi-phosphopeptides was proven. By controlling the treatment procedure, the partial dephosphorylation of multi-phosphopeptides was performed, and the multi-phosphorylated stees of the digest of beta-casein were found to be on serine residues at the possible sites of 17, 18 and 19.
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PMID:[Detection of multi-phosphopeptide sites using microcolumn high performance liquid chromatography-electrospray ionization tandem mass spectrometry]. 1797 96

The detection of phosphopeptides, especially multi-phosphopeptides, by tandem electrospray ionization mass spectrometry (ESI-MS/MS) is a great challenge due to their low abundance and the poor ionization efficiency of samples. In our recent study, a strategy was proposed for the analysis of trace multi-phosphopeptides which combined selective enrichment of phosphorylated peptides by TiO2 and dephosphorylation by alkaline phosphatase (AP). After separation by muHPLC, the profiles of enriched peptides before and after AP treatment were compared, and the additional peaks appearing in the latter case hinted at the existence of multi-phosphopeptides. Subsequently, an incomplete dephosphorylation reaction was performed to partially remove the phosphate groups so that the phosphorylation sites of the multi-phosphopeptides might be estimated. Through analysis of the digests of beta-casein and extracted proteins of bovine milk, more information on the multi-phosphopeptides was obtained by muHPLC-ESI-MS/MS than that obtained without AP treatment, which demonstrated that such a strategy might supply some potential information about trace multi-phosphopeptides lost in shotgun analysis.
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PMID:Characterization of multi-phosphopeptides by muHPLC-ESI-MS/MS with alkaline phosphatase treatment. 1821 Mar 78


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