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)

beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif (135)Asn-Gly(136). Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.
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PMID:Determination of the phosphorylation level and deamidation susceptibility of equine beta-casein. 1669 51

A new methodology for the preparation of enzyme-labeled protein polymers bearing pendent haptens was developed through the combination of chemical modification and posttranslational protein modification catalyzed by microbial transglutaminase (MTG). As a model hapten, trinitrobenzene (TNB) was chosen and chemically conjugated with the accessible Lys residues of beta-casein. The resultant trinitrophenylated beta-casein was further modified with formaldehyde to render the residual Lys residues inert toward self-cross-linking by MTG. Escherichia coli alkaline phosphatase (AP), comprising a specific peptide tag carrying a MTG-reactive Lys residue, was then conjugated to the Gln residues in beta-casein-TNB conjugates. The resultant AP-labeled beta-casein-bearing pendent TNB moieties (AP-betaCT) showed comparable specific activity with native AP. It was found that only the AP-betaCT with a sufficient number of pendent TNBs are capable of binding to a surface adsorbed with anti-TNP and anti-TNT antibodies, indicating the presence of polyvalent interactions. The utility of AP-betaCT was demonstrated by competitive immunoassays for trinitrophenol (TNP) and trinitrotoluene (TNT), with detection limits of 0.99 microg/L and 0.18 microg/L, respectively. The present study demonstrates the potential of dual labeling of protein scaffolds by chemical and enzymatic protein manipulation to create a new proteinaceous architecture.
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PMID:An enzyme-labeled protein polymer bearing pendent haptens. 1732 27

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

Covalent and site-specific protein immobilization catalyzed by microbial transglutaminase (MTG) was investigated using recombinant Escherichia coli alkaline phosphatase (AP) tagged with a glutamyl donor substrate peptide (MLAQGS) of MTG. A polystyrene surface physically coated with beta-casein or bovine serum albumin (BSA) was employed as an MTG-specific surface displaying reactive lysine residues. MTG-mediated protein immobilization through catalytic epsilon-(gamma-glutamyl)lysine bond formation between the peptide tag of recombinant APs and beta-casein- or BSA-coated surface was verified by the detection of AP activity on the surface. It was found that the length and the insertion position of the peptide tag did not significantly affect the efficacy of enzymatic immobilization of the recombinant APs. On the other hand, pH and ionic strength in the reaction media had crucial effects on the immobilization yields. Interestingly, the optimum pH range of MTG-mediated protein immobilization differed markedly from that for an MTG-catalyzed reaction in aqueous solution. The results suggest that the concentration of reactive species due to electrostatic interaction between the enzyme-substrate intermediate and the protein-adsorbed surface is a key factor governing MTG catalysis at a solid surface.
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PMID:Functional immobilization of recombinant alkaline phosphatases bearing a glutamyl donor substrate peptide of microbial transglutaminase. 1796 83

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

Herein, we describe three kinds of 2-DE using phosphate-affinity PAGE for the analysis of phosphoprotein isotypes. The first dimension is a urea-PAGE, IEF/NEPHGE, or SDS-PAGE, which are widely used. The second dimension is a phosphate-affinity SDS-PAGE using a phosphate-binding tag molecule, Phos-tag (Mn(2+)-Phos-tag SDS-PAGE). The first 2-D procedure coupling urea-PAGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separation of beta-casein phosphoisotypes. A typical protein sample containing multiple phosphoisotypes from beta-casein (with five phosphorylation sites) was prepared by partial dephosphorylation with alkaline phosphatase. The second procedure coupling IEF/NEPHGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separations of phosphoisotypes of caseins and in vitro kinase reaction products of Tau. The third procedure coupling normal SDS-PAGE and Mn(2+)-Phos-tag SDS-PAGE was applied to the separation of A431 cell lysates before and after stimulation with an epidermal growth factor. This procedure followed by immunoblotting with anti-mitogen-activated protein kinase(MAPK) and anti-Shc antibodies demonstrated the detection of phosphoisotypes in each protein isoform of MAPK1/2 (44 and 42 kDa) and Shc (66, 52, and 46 kDa) after the stimulation. By these novel 2-D procedures, the separations of phosphoprotein isotypes should be improved relative to those by current gel electrophoresis methods, including 1-D Mn(2+)-Phos-tag SDS-PAGE.
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PMID:Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes. 1915 64

Caseinophosphopeptides (CPP) are food mineral-rich components that may resist intestinal enzyme hydrolysis. We wondered whether phosphorylation and/or mineral binding induces resistance of CPP to intestinal hydrolysis. We used intestinal brush-border membrane vesicles to digest different forms of the beta-casein (1-25) peptide: unphosphorylated and phosphorylated carrier of varied cations. The results showed that the activity of alkaline phosphatase seems not to be specific to either the phosphorylation degree or the phosphorylation sites whereas phosphorylations limited the action of peptidases. Studying the mechanism and the kinetics of hydrolysis of the different peptides allows understanding how some cations prevent more CPP from hydrolysis than others. The action of both exo- and endopeptidases was limited for the beta-CN (1-25) peptide bound to zinc or copper. Actually the peptide bound to copper was almost not hydrolyzed during the digestion, suggesting that coordination bond of copper to CPP inhibits the action of both phosphatase and peptidases.
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PMID:Phosphorylation and coordination bond of mineral inhibit the hydrolysis of the beta-casein (1-25) peptide by intestinal brush-border membrane enzymes. 2051


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