Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

In this study, gas chromatography/mass spectrometry revealed the presence of stoichiometric amounts of myo-inositol in association with serum corticosteroid-induced isozyme of alkaline phosphatase (CALP) in canine serum. Such remnants are consistent with prior membrane attachment of serum CALP and its release into serum by endogenous phospholipase activity. Serum CALP was further shown to behave similarly to CALP released from hepatocyte membranes by glycosyl phosphatidylinositol phospholipase D (GPI-PLD) and differently from CALP solubilized by GPI-phospholipase C (PLC) on both native polyacrylamide gel electrophoresis and Western blot analysis using anti-cross-reacting determinant antibody. In addition to bile canalicular surfaces, CALP activity was found over hepatocyte sinusoidal surfaces by histochemical staining of canine liver sections. A significantly higher ratio of CALP to total alkaline phosphatase activity was observed in serum as opposed to bile in 10 of 11 paired serum and bile samples from dogs. This suggested that bile is not likely to be the source of serum CALP and is consistent with the release of CALP from hepatocyte basolateral surfaces directly into serum. It was concluded that serum CALP was once membrane bound and was released by phospholipase activity into serum. Our findings are consistent with release of CALP from the sinusoidal surfaces of hepatocytes into serum either by endogenous GPI-PLD activity or release by GPI-PLC followed by modification of the phosphatidylinositol remnant in vivo.
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PMID:Canine corticosteroid-induced alkaline phosphatase in serum was solubilized by phospholipase activity in vivo. 765 69

Treatment of liver plasma membranes with Triton X-100 allowed an endogenous alkaline phosphatase-converting activity to convert amphiphilic alkaline phosphatase (membrane anchor covalently attached) to hydrophilic dimers that resemble the enzyme found in normal plasma. The Triton-solubilized activity was unaffected by protease inhibitors. Amphiphilic alkaline phosphatase purified from human liver and placenta were both substrates. The Triton-solubilized enzyme would not hydrolyze L-3-phosphatidyl(2-3H)-inositol or p-nitrophenylphosphoryl choline, nor would it cleave endogenous alkaline phosphatase from intact plasma membranes. These observations and the analysis of the protein product of the hydrolysis of placental alkaline phosphatase, following treatment with the converting activity, indicated that the enzyme has the specificity of a glycosyl-phosphatidylinositol phospholipase D. Further characterization of the enzyme activity suggests additional similarities with the glycosyl-phosphatidylinositol phospholipase D found in mammalian plasma. Alkaline phosphatase-converting activity in plasma membranes represented the same percent of total protein as it did in whole liver, whereas serum contained 3- to 10-times this amount. Endogenous converting activity in plasma membranes was not solubilized by salt washes, sonication, or repeated freeze-thaw treatments. We believe it is unlikely that the alkaline phosphatase-converting activity in liver plasma membranes resulted from adsorption of the enzyme present in plasma.
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PMID:Human liver plasma membranes contain an enzyme activity that removes membrane anchor from alkaline phosphatase and converts it to a plasma-like form. 820 Jan 15

A method is described for large-scale purification of glycosylphosphatidylinositol-anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acid/mol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components. No significant differences between native glycosylPtdIns-anchored and hydrophilic alkaline phosphatases from both sources were found regarding Km, Vmax, the type of inhibition and inhibition constants of the amino acids L-leucine, L-phenylalanine, and L-tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively. Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl-Sepharose chromatography. Fraction I corresponds to the anchorless enzyme, fractions II-V differ in their susceptibility to phospholipases. Fractions II and IV are completely split by PtdIns-PLC or PLD action, almost 50% of fraction III is split by PtdIns-PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions II, III, IV, and V, respectively. Aggregates of glycosylPtdIns-anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X-100. By gel chromatography in the presence of Brij 35, fractions II-V form stable multiple aggregates of dimers and may bind different amounts of the detergent. These data, together with fatty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction III is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns-anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns-PLC. The similarity of all measured parameters of both enzymes suggests that the glycosylPtdIns-anchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.
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PMID:Heterogeneity of glycosylphosphatidylinositol-anchored alkaline phosphatase of calf intestine. 822 55

A reproducible substrate for the assay of phosphatidylinositol-specific phospholipase D (PIPLD) can be prepared by extracting alkaline phosphatase from placental tissue with n-butanol under alkaline conditions. The alkaline phosphatase thus prepared retains its hydrophobic glycan phosphatidylinositol (GPI) anchor and aggregates into high M(r) forms. Incubation with serum hydrolyses the phosphate inositol linkage by PIPLD action, producing a less lipophilic, non-aggregated isoform of alkaline phosphatase. Three methods of measuring the amount of this isoform produced after a timed incubation with serum are described and compared: two types of phase partitioning systems, and electrophoresis and densitometry of the products after gradient-pore electrophoresis. All give comparable and reproducible measurements of PIPLD; however, the electrophoretic method is preferred for routine analysis.
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PMID:A method for the assay of phosphatidylinositol-specific phospholipase D activity in serum. 840 31

This study investigated ectoenzyme release from small intestine brush border membranes (duodenum and jejunum, Preparation A; ileum, Preparation B) of mice by the action of phosphatidylinositol-specific phospholipase C or glycosyl-phosphatidylinositol-specific phospholipase D. Most of the alkaline phosphatase was solubilized from Preparation A, but about 60% was released from Preparation B. As for alkaline phosphodiesterase I activity, 15 and 10% were released from Preparations A and B, respectively. With Preparation B, octylglucoside treatment followed by phosphatidylinositol-specific phospholipase C or glycosyl-phosphatidylinositol-specific phospholipase D completely solubilized the alkaline phosphatase activity. However, this treatment did not change the ratio of release of alkaline phosphodiesterase I from Preparation A or B. These results indicate that the resistance to alkaline phosphatase found in Preparation B is due to hindered accessibility of the bonding splitting by phosphatidylinositol-specific phospholipase C and not to a modified glycosyl-phosphatidylinositolanchor.
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PMID:Release of ectoenzymes from small intestine brush border membranes of mice by phospholipases. 905 73

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is a secretory protein present in high amounts in mammalian body fluids. Its cDNA has been isolated and encodes a signal peptide of 23 amino acids and the mature protein of 816 amino acids. We generated cDNAs encoding a signal peptide-deficient and a GPI-anchored form of GPI-PLD and transiently transfected these constructs into COS-1 cells. The signal peptide-deficient form of GPI-PLD was expressed as a 90-kDa protein that was catalytically active and was localized intracellularly. Cells transfected with cDNA encoding the GPI-anchored form of GPI-PLD expressed a catalytically active enzyme of 100 kDa that could be labelled with [3H]ethanolamine demonstrating its modification by a GPI structure. Expression of the GPI-anchored form of GPI-PLD resulted in the release of endogenous GPI-anchored alkaline phosphatase from COS-1 cells, whereas expression of the intracellular form of GPI-PLD had no effect on membrane attachment of endogenous alkaline phosphatase. Similarly, in cells cotransfected with GPI-anchored placental alkaline phosphatase (PLAP) and the GPI-anchored form of GPI-PLD, PLAP was released into the cell culture supernatant while expression of the signal peptide-deficient form of GPI-PLD did not affect the amount of cell-associated PLAP.
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PMID:Expression of intracellular and GPI-anchored forms of GPI-specific phospholipase D in COS-1 cells. 926 57

The enzymatic properties of glycosylphosphatidylinositol-specific phospholipase D (EC 3.1.4.50) were characterized using a 6,000-fold purified enzyme. This was obtained in 100 microg amounts from human serum with a recovery of 35%. Pure alkaline phosphatase containing one anchor moiety per molecule was used as substrate. The enzyme is stimulated by n-butanol, but in contrast to other phospholipases this activation is not produced by a transphosphatidylation reaction. The previously reported non-linearity of the specific activity with respect to phospholipase concentration in the test was no longer observed upon purification, indicating inhibitor removal. The serum inhibitor(s) co-chromatograph with serum proteins and lipoproteins. The main part of the inhibitory activity was found in the lipid fraction after protein denaturation and can be subfractionated into acid phospholipids, cholesteryl esters and triacylglycerides. Added phosphatidyl-serine, phosphatidylinositol, phosphatidylglycerol, gangliosides, cholesteryl esters, and sphingomyelins turned out to be strong inhibitors, as well as phosphatidic acid. Phosphatidylethanolamine and various monoacylglycerols were found to be activators. The low glycosylphosphatidylinositol-specific phospholipase activity found in native serum did not increase significantly upon 90% removal of phospholipids by n-butanol. High serum concentrations of strongly inhibiting compounds, complex kinetic interactions among aggregates of these substances, and compartmentalization effects are discussed as possible reasons for the observed inactivity.
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PMID:Glycosylphosphatidylinositol-specific phospholipase D of human serum--activity modulation by naturally occurring amphiphiles. 1093 80

Mammalian glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is capable of releasing GPI-anchored proteins by cleavage of the GPI moiety. A previous study indicated that overexpression of GPI-PLD in mouse RAW 264.7 monocytes/macrophages could be cytotoxic, since survivors of stable transfections had enzymic activity no higher than untransfected cells [Du and Low (2001) Infect. Immun. 69, 3214-3223]. We investigated this phenomenon by transfecting bovine GPI-PLD cDNA stably into Chinese hamster ovary (CHO) cells using a bi-cistronic expression system. The surviving transfectants showed an unchanged cellular level of GPI-PLD, supporting the cytotoxicity hypothesis. However, when using a CHO mutant defective in the second step of GPI biosynthesis as host, the expression level of GPI-PLD in stable transfectants was increased by 2.5-fold compared with untransfected or empty-vector-transfected cells. To identify the mechanism, we studied another CHO cell mutant (G9PLAP.D5), which seems to be defective at a later stage in GPI biosynthesis. In sharp contrast with wild-type cells, GPI-PLD activity in G9PLAP.D5 transfected with bovine GPI-PLD cDNA was 100-fold higher than untransfected or empty-vector-transfected cells. This was accompanied by a significant release of alkaline phosphatase into the medium and a decrease in membrane-associated alkaline phosphatase. Taken together, our results indicate that overexpression of GPI-PLD is lethal to wild-type cells, possibly by catalysing the overproduction of GPI-derived toxic substances. We propose that cells with abnormal GPI biosynthesis/processing can escape the toxic effect of these substances.
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PMID:Tolerance of glycosylphosphatidylinositol (GPI)-specific phospholipase D overexpression by Chinese hamster ovary cell mutants with aberrant GPI biosynthesis. 1174 35