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 pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10-1 for rho-nitrophenylphosphate, 8-6 for phosphoserine, 8-0 for phosvitin and 6-8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of alphas- and beta-caseins or of alphas-, beta-and kappa-caseins and micellar casein. Mixtures of 2 components involving kappa-casein were more readily dephosphorylated than alphas- and beta-casein mixtures. At pH 6-8, lactose, whey proteins and phosphate ions had an inhibitory effect. beta-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described.
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PMID:Dephosphorylation of bovine casein by milk alkaline phosphatase. 0 76

Three peptides have been formed by proteolytic digestion of individual casein proteins and their secondary structures characterised by far-UV circular dichroism (CD). Peptide alpha s1(1-23), residues 1-23 of alpha s1-casein, was generated by treatment of the parent protein with chymosin. Peptides beta(1-28) and beta(1-52), residues 1-28 and 1-52 of beta-casein, were plasmin- and chymotrypsin-generated fragments, respectively. Analysis of the CD spectra revealed that in aqueous solution all three peptides have secondary structures composed exclusively of beta-sheet and random coil. A limited amount of alpha-helix was formed in two of the three peptides upon treatment with high concentrations (greater than 40% (v/v] of 2,2,2-trifluoroethanol. Partial dephosphorylation (60%) of beta(1-28) and beta(1-52) by treatment with alkaline phosphatase resulted in homogeneous preparations, as judged by polyacrylamide gel electrophoresis, which exhibited increased hydrophobicity. This reduction in the level of phosphorylation of serine residues 15, 17, 18 and 19 led to increased propensity for helix formation in the peptides in the presence of 2,2,2-trifluoroethanol, but no alpha-helical structures were detected in the dephosphorylated peptides in the absence of 2,2,2-trifluoroethanol.
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PMID:The secondary structure of peptides derived from caseins: a circular dichroism study. 316 68

First lactation milk yield and percentages of fat, solids-not-fat, and protein were analyzed in Guernsey cows to determine relationships of production traits to genetic markers at 16 polymorphic loci. The polymorphic systems examined were blood groups A, B, C, F, J, L, M, S, and Z; blood proteins transferrin, hemoglobin, and alkaline phosphatase; and milk proteins beta-lactoglobulin, alpha s1-casein, beta-casein, and kappa-casein. Different statistical models were utilized to evaluate direct genetic marker effects, linkage group effects, and heterozygosity effects. There were many indications of relationship with milk composition traits but for milk yield only for A system direct effects and for F system linkage effects. Pronounced associations between markers and component percentages were noted for the J, Z, and beta-lactoglobulin systems with fat percentage, for the M system with fat and solids-not-fat percentages, for the alkaline phosphatase system with solids-not-fat percentage and protein percentages, and for L and alpha s1-casein systems with protein percentage. Additionally, the interaction of beta-lactoglobulin and alpha s1-casein markers was significant for deviations of percent fat and percent solids-not-fat.
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PMID:Associations of bovine blood and milk polymorphisms with lactation traits: Guernseys. 344 10

Sequential probability tests were employed to examine genetic linkage among, cattle A, B, C, F, J, L, M, S, Z, and R' blood group loci, the hemoglobin locus, the serum transferrin, amylase, and alkaline phosphatase loci, and the milk protein loci beta-lactoglobulin, alpha S1-casein, beta-casein, and kappa-casein. Linkage was evident between the A and hemoglobin loci, the J and beta-lactoglobulin loci, the alpha S1-casein and beta-casein loci, the alpha S1-casein and kappa-casein loci, and the beta-casein and kappa-casein loci. Recombination fractions for these respective combinations were 0, .18, .03, .04, and .06. There was a suggestion of possible loose linkage (recombination fraction = .4) between the beta-lactoglobulin locus and the casein complex. Linkage could be excluded for most other combinations at .25 recombination.
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PMID:Linkage relationships among loci of polymorphisms in blood and milk of cattle. 726 22

A rapid, picomole-scale method is described to locate phosphorylation sites in phosphoproteins by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with enzymatic modification of the analyte. There are three steps to locate phosphorylation sites in a phosphoprotein: (i) degradation of the phosphoprotein into small peptides by specific enzymatic or chemical reactions; (ii) identification of the phosphopeptides by -80 (or multiples of -80)-Da mass shifts in the mass spectra after dephosphorylation with alkaline phosphatase; (iii) location of the phosphorylation sites by mass mapping. As the size of the protein increases, it is advantageous to fractionate the mixture by HPLC and analyze each fraction by MALDI-TOF-MS. To perform mass mapping, the primary structure of the protein must be known. Bovine beta-casein was analyzed by this method. The conclusions about the specific phosphorylation sites of bovine beta-casein from our data coincide with previously reported results. From calculations, it is found that a mass spectrometer with 0.1% mass accuracy is sufficient, for mass mapping, to identify completely or partially digested tryptic peptides in the mass range of 100-8000 Da from bovine beta-casein (MW 23,983).
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PMID:An approach to locate phosphorylation sites in a phosphoprotein: mass mapping by combining specific enzymatic degradation with matrix-assisted laser desorption/ionization mass spectrometry. 805 60

We have shown that the cellular mechanisms of the mammary gland can be used to produce a phosphorylated form of a normally unphosphorylated milk protein. This was achieved by the insertion of a beta-casein DNA sequence coding for a group of mammary gland casein kinase recognition sites into ovine beta-lactoglobulin. Transgenic mice carrying this modified gene were generated and lactating females were shown to produce a novel beta-lactoglobulin in their milk. The infrared spectrum, reactivity to antiphosphoserine antibody and reduction of electrophoretic mobility on treatment with alkaline phosphatase showed that the novel protein recovered from the milk whey (serum) was phosphorylated and molecular mass determination by mass spectrometry was consistent with the phosphorylation of one or two residues. A similar level of phosphorylation was measured by quantitative infrared spectroscopy. Centrifugation of the milk to pellet the casein micelles showed that most of the phosphorylated beta-lactoglobulin was in the whey and hence not incorporated into casein micelles.
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PMID:Insertion of a casein kinase recognition sequence induces phosphorylation of ovine beta-lactoglobulin in transgenic mice. 1023 27

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins is required to fully understand protein function and regulatory events in cells and organisms. Here we present a mass spectrometry (MS) based experimental strategy for the identification and mapping of phosphorylation site(s) using only low-picomole amounts of phosphoprotein starting material. Miniaturized sample preparation methods for MS facilitated localization of phosphorylation sites in phosphoproteins isolated by polyacrylamide gel electrophoresis. Custom made, nanoscale immobilized Fe(III) affinity chromatography (Fe(III)-IMAC) columns were employed for enrichment of phosphorylated peptides from crude peptide mixtures prior to off-line analysis by matrix-assisted laser desorption/ionization (MALDI) MS or nanoelectrospray tandem mass spectrometry (MS/MS). An optimized and sensitive procedure for alkaline phosphatase treatment of peptide mixtures was implemented, which in combination with nano-scale Fe(III)-IMAC and MALDI-MS allowed unambiguous identification of phosphopeptides by observation of 80 Da mass shifts. Nanoelectrospray MS/MS was used for phosphopeptide sequencing for exact determination of phosphorylation sites. The advantages and limitations of the experimental strategy was demonstrated by enrichment, identification and sequencing of phosphopeptides from the model proteins ovalbumin and bovine beta-casein isolated by gel electrophoresis. Furthermore, an autophosphorylation site at Ser-3 in recombinant human casein kinase-2 beta subunit was determined. The potential of miniaturized Fe(III)-IMAC and MALDI-MS for characterization of in vivo phosphorylated proteins was demonstrated by identification of tryptic phosphopeptides derived from the human p47/phox phosphoprotein isolated by two-dimensional gel electrophoresis.
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PMID:Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis. 1168 Aug 68

A novel disposable high-density matrix assisted laser desorption/ionization (MALDI) target plate made either from polymethylmethacrylate (PMMA) or polycarbonate (PC) is presented where thousands (1,200-1,600) of samples can be deposited and subsequently analyzed by MALDI-time of flight (TOF) mass spectrometry. Good reproducibility was obtained across the plate regardless of position on the target plate with a relative standard deviation (RSD) on the peak intensity of typically 30% calculated from data generated by analysis of a 10 nm peptide mixture of angiotensin I, II, III and bradykinin. The nanovial array format combined with microdispensing technology makes it possible to carry out in-vial chemistry on deposited samples. This is demonstrated by the analysis of peptides from beta-casein and subsequent in-vial dephosphorylation of its phosphopeptides at 10 fmol levels by microdispensing of alkaline phosphatase, into the nanovial. The mass spectra obtained from these polymeric targets provides can also be used in high sensitivity applications as shown by peptide mass fingerprinting of human fibroblast proteins separated by two-dimensional gel electrophoresis.
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PMID:Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: II. Biological applications. 1170 Jul 30

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.
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PMID:Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase. 1600 75

Clusters of phosphoserine residues in cow milk caseins bind iron (Fe) with high affinity. Casein inhibits Fe absorption in humans, but protein hydrolysis lessens this effect. Phosphopeptides from different caseins gave conflicting results on Fe absorption; release of phosphate residues by intestinal alkaline phosphatase could be a key point of that metabolism. The objectives of this study were to compare the absorption of Fe complexed to caseinophosphopeptides (CPP) of the main cow milk caseins beta-casein (beta-CPP) and alpha(s)-caseins (alpha(s1)-CPP) and to assess the role of alkaline phosphatase on this absorption. Two experimental models were used: an in vivo perfused rat intestinal loop and an in vitro Caco-2 cell culture model. In addition, we determined the effect of an intestinal phosphatase inhibitor on these various forms of Fe. Gluconate Fe was used as control. In both models, uptake and net absorption of Fe complexed to CPP from alpha(S1)-caseins were significantly lower than from Fe complexed to beta-CPP. Inhibition of the intestinal phosphatase significantly increased the uptake and the absorption of Fe complexed to beta-CPP without effect on the other forms of Fe. These results confirm the enhancing effect of beta-casein and its CPP on Fe absorption. The differences between CPP could be explained by their structural and/or conformational features: binding Fe to alpha(S1)-CPP could impair access to digestive enzymes, whereas beta-CPP-bound Fe is better absorbed than its free form. The differences in protein composition between cow and breast milk, which does not contain alpha-casein, could explain some of their differences in Fe bioavailability.
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PMID:Milk proteins and iron absorption: contrasting effects of different caseinophosphopeptides. 1618 1


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