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)

A phosphatidylinositol phosphodiesterase from the culture broth of Bacillus cereus, was purified to a homogeneous state as indicated by polyacrylamide gel electrophoresis, by ammonium sulfate precipitation and chromatography with DEAE-cellulose and CM-Sephadex. The enzyme (molecular weight: 29000 +/- 1000) was maximally active at pH 7.2-7.5, AND NOT INFLUENCED BY EDTA, ophenanthroline, monoiodoacetate, p-chloromercuribenzoate or reduced glutathione. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, under the conditions examined. The products from phosphatidylinositol of enzyme reaction were diacylglycerols and a mixture of myoinositol 1- and 1, 2-cyclic phosphates, suggesting that the enzyme was a phosphatidylinositol-specific phospholipase C. The enzyme released alkaline phosphatase quantitatively from rat kidney slices. A kinetic analysis was made on the release of alkaline phosphatase. The results suggest that phosphatidylinositol-specific phospholipase C can specifically act on plasma membrane of rat kidney slices.
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PMID:Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity. 1 Sep 86

The release of plasma membrane ecto-enzymes by a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was investigated. There was no effect on L-leucyl-beta-naphthylamidase, alkaline phosphodeisterase I and Ca2+- or MG2+-ATPase, but substantial proportions of the alkaline phosphatase and 5-nucleotidase were released. There was no simultaneous release of phospholipid and the solubilized enzymes were not exluded from Sepharose 6-B. It was therefore concluded that release was not a secondary consequence of membrane vesiculation but occurred as a result of the disruption of specific interactions involving the phosphatidylinositol molecule.
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PMID:Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C. 20 48

Purified phosphatidylinositol-specific phospholipase C from Staphylococcus aureus released a substantial proportion of the total alkaline phosphatase activity from a wide range of tissues from several mammalian species. Co-purification of the phospholipase C and alkaline phosphatase-releasing activities and the inhibition of both these activities by iso-osmotic salt solutions suggested that the releasing effect was unlikely to be due to a contaminant.
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PMID:Release of alkaline phosphatase from membranes by a phosphatidylinositol-specific phospholipase C. 58 58

A subcellular fractionation method to isolate simultaneously apical and basolateral plasma membrane fractions from the human adenocarcinoma cell line Caco-2, grown on filter supports, is described. The method employs sucrose-density-gradient centrifugation and differential precipitation. The apical membrane fraction was enriched 14-fold in sucrase-isomaltase and 21-fold in 5'-nucleotidase compared with the homogenate. The basolateral membrane fraction was enriched 20-fold relative to the homogenate in K(+)-stimulated p-nitrophenylphosphatase. Alkaline phosphatase was enriched 15-fold in the apical membrane fraction and 3-fold in the basolateral membrane fraction. Analytical density-gradient centrifugation showed that this enzyme was a true constituent of both fractions, and experiments measuring alkaline phosphatase release following treatment with phosphatidylinositol-specific phospholipase C showed that in both membrane fractions the enzyme was glycosyl-phosphatidylinositol-linked. There was very little contamination of either membrane fraction by marker enzymes of the Golgi complex, mitochondria or lysosomes. Both membrane fractions were greater than 10-fold purified with respect to the endoplasmic reticulum marker enzyme alpha-glucosidase. Protein composition analysis of purified plasma membrane fractions together with domain-specific cell surface biotinylation experiments revealed the presence of both common and unique integral membrane proteins in each plasma membrane domain. The post-synthetic transport of endogenous integral plasma membrane proteins was examined using the devised subcellular fractionation procedure in conjunction with pulse-chase labelling experiments and immunoprecipitation. Five common integral membrane proteins immunoprecipitated by an antiserum raised against a detergent extract of the apical plasma membrane fraction were delivered with the same time course to each cell-surface domain.
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PMID:The post-synthetic sorting of endogenous membrane proteins examined by the simultaneous purification of apical and basolateral plasma membrane fractions from Caco-2 cells. 131 18

Saccharomyces cerevisiae contains an amphiphilic cAMP-binding glycoprotein at the outer face of the plasma membrane (M(r) = 54,000). It is converted to a hydrophilic form by treatment with glycosyl-phosphatidylinositol-specific phospholipases C and D (GPI-PLC/D), suggesting membrane anchorage by a covalently bound glycolipid. Determination of the constituents of the purified anchor by gas-liquid chromatography and amino acid analysis reveals the presence of glycerol, myo-inositol, glucosamine, galactose, mannose, ethanolamine, and asparagine (as the carboxyl-terminal amino acid of the Pronase-digested protein to which the anchor is attached). Complementary results are obtained by metabolic labeling, indicating that fatty acids and phosphorus are additional anchor constituents. The phosphorus is resistant to alkaline phosphatase, whereas approximately half is lost from the protein after treatment with GPI-PLD or nitrous acid, and all is removed by aqueous HF indicating the presence of two phosphodiester bonds. Inhibition of N-glycosylation by tunicamycin or removal of protein-bound glycan chains by N-glycanase or Pronase does not abolish radiolabeling of the anchor structure by any of the above compounds. Analysis of the products obtained after sequential enzymic and chemical degradation of the anchor agrees with the arrangement of constituents in GPIs from higher eucaryotes. Evidence for anchorage of the yeast cAMP-binding protein by a GPI anchor is strengthened additionally by the reactivity of the GPI-PLC-cleaved anchor with antibodies directed against the cross-reacting determinant of trypanosomal variant surface glycoproteins.
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PMID:The cAMP-binding ectoprotein from Saccharomyces cerevisiae is membrane-anchored by glycosyl-phosphatidylinositol. 133 92

1. Alkaline phosphatase is covalently bound to bovine mammary microsomal membranes and milk fat globule membranes through linkage to phosphatidylinositol as demonstrated by the release of alkaline phosphatase following treatment with phosphatidylinositol-specific phospholipase C. 2. The release of alkaline phosphatase from the pellet to the supernatant was demonstrated by enzyme assays and electrophoresis. 3. Electrophoresis of the solubilized enzymes showed that the alkaline phosphatase of the microsomal membranes contained several isozymes, while only one band with alkaline phosphatase activity was seen in the fat globule membrane. 4. Levamisole and homoarginine were potent inhibitors of the alkaline phosphatase activities in both membrane preparations and in bovine liver alkaline phosphatase, but not in calf intestinal alkaline phosphatase.
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PMID:Alkaline phosphatase in the lactating bovine mammary gland and the milk fat globule membrane. Release by phosphatidylinositol-specific phospholipase C. 137 15

An acidic glycoconjugate could be extracted from a delipidated residue fraction of [3H]galactose, [3H]mannose or [32P]orthophosphate metabolically labeled Entamoeba histolytica with water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017). The radioactively labeled glycoconjugate comprised 50-55% of the total [3H]galactose label incorporated into macromolecules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the radiolabeled glycoconjugate showed two diffuse smears centering around 110 kDa and 45 kDa. Similar profiles were observed for both [3H]galactose- and [32P]orthophosphate-labeled glycoconjugate. No such bands were visible in [35S]methionine-labeled material. The hydrophobic nature of this glycoconjugate was inferred from its chromatographic behavior on phenyl-Sepharose. The molecule was rendered hydrophilic after digestion with phosphatidylinositol-specific phospholipase C. It was also sensitive to deamination by nitrous acid. Mild acid hydrolysis led to its fragmentation into smaller molecules as revealed by Sepharose 4B chromatography. Paper chromatographic analysis of the depolymerized [3H]galactose- and [3H]mannose-labeled fragments revealed that each was sensitive to alkaline phosphatase. The major dephosphorylated fragment migrated as an apparent galactose and mannose containing disaccharide which migrated identically to the Gal beta 1-4Man disaccharide derived from the lipophosphoglycan of Leishmania donovani. The above data support the existence of a major acidic glycoconjugate in E. histolytica bearing striking structural similarities to the lipophosphoglycan of Leishmania.
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PMID:Identification and partial characterization of a lipophosphoglycan from a pathogenic strain of Entamoeba histolytica. 147 94

1. Considerable amounts of intestinal alkaline phosphatase (AP) were found intralumenally in all animal species investigated, i.e. calf, pig, goat, rat, mouse, guinea pig, hen and carp. The ratios between the total activity of AP found intralumenally and the total intestinal activity vary considerably. Calves and pigs show the highest, i.e. 0.77 and 0.44, respectively, while rodents have much lower ratios. Only 20-34% of the intralumenal alkaline phosphatase (IAP) of the calf and pig is soluble and not within the sediment after centrifugation at 135,000 x g for 60 min. whereas the IAP of rodents is soluble in the range of 60-72% of the total IAP. 2. For the IAP of the mucosa and chyme of calf, all criteria were found which are generally used, indicating a glycosylphosphatidylinositol (GlcPtdIns) anchor as proved by strong hydrophobicity using Triton X-114 phase partitioning, phenyl-Sepharose binding and enzyme aggregation, and the susceptibility to phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and papain digestion. 3. More than 80% of the mucosa alkaline phosphatase (MAP) of the proximal part of the intestine and of the particulate fraction of IAP exhibit these criteria indicating the presence of the GlcPtdIns-anchor structure, whereas the anchor content of the soluble intralumenal enzyme decreases from the pylorus to the ileocecal junction. 4. MAP partially purified to a specific activity of 1747 IU/mg retains the anchor structure. 5. The results presented indicate that the release of large amounts of AP into the chyme is realized without splitting the GlcPtdIns anchor. The possible intralumenal function of this form of AP is discussed.
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PMID:Evidence for glycosylphosphatidylinositol anchoring of intralumenal alkaline phosphatase of the calf intestine. 164 47

Recently we reported that the expression of the enzyme alkaline phosphatase (APase) is a marker for B cell activation. Enzymatic activity was found only in activated B cells and not T cells. Using flow cytometry we showed that some of the APase was found on the cell membranes (mAPase) and by functional assays, some was spontaneously released into the tissue culture medium. In the present report the expression of mAPase on activated B lymphocytes is more fully characterized. Two mAb specific for rat APase were used to measure the kinetics of the membrane expression of mAPase. Within 48 h of activation, mAPase is detected by flow cytometry and increases coordinately with both the transferrin receptor and IL-2R. Maximal membrane expression of mAPase in terms of number of positive cells and mean fluorescent intensity, is detected by day 4 to 5 of culture. Using hydroxyurea and demecolcine to block cells at G1/S and G2/M, respectively, it appeared that the initial expression of mAPase occurred as cells progressed into S phase of the cell cycle. This was confirmed using two-color flow cytometric analysis with the Hoechst DNA stain 33342 and the FITC-labeled APase-specific mAb. Finally, using phosphatidylinositol-specific phospholipase C we were able to show that 60 to 80% of the mAPase is linked to the membrane via a glycosyl-phosphatidylinositol linkage. From this we have concluded that mAPase can be added to a growing list of glycoproteins that are anchored to the membrane by the glycosyl-phosphatidylinositol linkage and are expressed on differentiating B cells. This list now includes Thy-1, BLAST-1, Jlld, and mAPase.
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PMID:Alkaline phosphatase on activated B cells characterization of the expression of alkaline phosphatase on activated B cells. Kinetics and membrane anchor. 165 49

The construction of four vectors for high-level expression in Escherichia coli of the phosphatidylinositol-specific phospholipase C from Bacillus cereus or Bacillus thuringiensis is described. In all constructs the coding sequence for the mature phospholipase is precisely fused to the E. coli heat-stable enterotoxin II signal sequence for targeting of the protein to the periplasm. In one set of plasmids expression of the B. cereus or B. thuringiensis enzyme is under control of the E. coli alkaline phosphatase promoter, while in a second set of plasmids expression is under control of a lac-tac-tac triple tandem promoter. A simple and rapid procedure for complete purification of the phospholipase C overproduced in E. coli, involving isolation of the periplasmic proteins by osmotic shock followed by a single column chromatography step, is described. The largest quantity of purified enzyme, 40-60 mg per liter culture, is obtained with the plasmid expressing the B. cereus enzyme under control of the lac-tac-tac promoter. Lower quantities are obtained with the plasmids containing the alkaline phosphatase promoter (15-20 and 4-6 mg/liter for the B. cereus and B. thuringiensis enzymes, respectively) and with the plasmid expressing the B. thuringiensis phospholipase under control of the lac-tac-tac promoter (15-20 mg/liter). A comparison of the functional properties of the recombinant phospholipases with the native enzymes isolated from B. cereus or B. thuringiensis culture supernatant shows that they are identical with respect to their catalytic functions, viz., cleavage of phosphatidylinositol and cleavage of the glycosyl-phosphatidylinositol membrane anchor of bovine erythrocyte acetylcholinesterase.
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PMID:High-level expression in Escherichia coli and rapid purification of phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringiensis. 166 69


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