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)

Soluble alkaline phosphatase from Thiobacillus thioparus cells was purified about 230-fold. The enzyme had a mol. wt. of 50 000 daltons, optimum pH at 10.5, and was heat-resistant in the presence of diethanolamine. Polyacrylamide-gel electrophoresis demonstrated contamination of the preparation with inactive proteins and the presence of two active bands. The enzyme activity was distinctly stimulated by increasing concentrations of Tris or diethanolamine. In the presence of glycine, 1 mM-Zn2+ enhanced the enzyme activity; in Tris or diethanolamine buffers the activity was stimulated by 1 mM-Mg2+ whereas Zn2+ had a strong inhibitory effect. Glycine at concentrations exceeding 25 mM also inhibited the enzyme. Specificity of the enzyme is fairly broad.
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PMID:Alkaline phosphatase of Thiobacillus thioparus. Partial purification and properties of the enzyme. 0 90

A brush border preparation from rat intestine was incubated with rat intrinsic factor-vitamin B12 complex in 0.01 M Tris-HCl buffer, pH 7.4. The 57Co-B12 uptake to brush borders was proportional to the amount of protein or to alkaline phosphatase activity in the preparations. The uptake increased with time of incubation. At 37degreesC, the uptake after incubation for 15 min was 80-85% of that for one hr. The uptake at 4degreesC was approximately 70% of that at 37degreesC. Ther was no difference as a result of adding glucose to the incubation medium. The uptake was observed in the alkaline environment above pH 6.3. Maximum uptake occurred at pH 8.0. Brush borders washed with Krebs-Ringer bicarbonate buffer (pH 7.4) exhibited no difference in B12 uptake, whether in the presence or absence of calcium ion. But brush borders washed with ethylenediaminetetraacetate exhibited no uptake when incubated in calcium-free medium. The uptake reached a maximum by addition of calcium ion at a concentration of 0.3 mM, and was not alter up to 10 mM. Addition of magnesium ion exhibited no uptake. Calcium-dependent B12 uptake was markedly inhibited by manganese ion. Magnesium ion seemed to slightly inhibit the calcium-dependent uptake.
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PMID:Effects of divalent cations on vitamin B12 adsorption to brush borders of rat intestine. 0 95

Calf pancreas microsomes incorporated radioactive D-mannose from GDP-D-[14C]mannose into lipid-bound oligosaccharides extracted with chloroform/methanol/water (10/10/2.5, v/v). Several products, which probably differed in the size of the oligosaccharide moiety, were labeled. These could be partially resolved by thin layer chromatography and DEAE-cellulose chromatography. The labeled lipid-bound oligosaccharides were retained on DEAE-cellulose more strongly than synthetic dolichyl alpha-D-[14C]mannopyranosyl phosphate. They were stable to mild alkali, but labile to acid and hot alkali. Acid treatment yielded a neutral 14C-labeled oligosaccharide fraction which was estimated by gel filtration to have a minimum of 8 monosaccharide residues. Hot alkali treatment yielded a mixture of neutral and acidic 14C-labeled oligosaccharides which could be transformed into neutral products by alkaline phosphatase. The D-[14C]mannose residues were alpha-linked at the nonreducing terminus of the oligosaccharides since they could be removed completely with alpha-mannosidase. Most of the D-[14C]mannose-labeled oligosaccharides were retained on concanavalin A Sepharose and eluted with methyl alpha-D-mannopyranoside. Pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate incubated with calf pancreas microsomes in the presence of sodium taurocholate was efficiently utilized as donor of alpha-D-mannosyl residues in lipid-bound oligosaccharides. The products formed from dolichyl beta-D-[14C]mannopyranosyl phosphate were identical with those formed from GDP-D-[14C]mannose, and evidence was obtained to show that the dolichyl beta-D-[14C]mannopyranosyl phosphate was serving as donor without prior conversion to GDP-D-[14C]mannose. Transfer of mannose from dolichyl beta-D-[14C]mannopyranosyl phosphate to lipid-bound oligosaccharides took place at a pH optimum of 7.3, whereas transfer to the precipitate containing glycoproteins was greatest at pH 6.0 in Tris/maleate buffer. The addition of divalent cation was not required, but low concentrations of EDTA were extremely inhibitory. The carbohydrate composition of the lipid-bound oligosaccharides of microsomal membranes was investigated by gas-liquid chromatography and by reduction with sodium borotritide. A heterogeneous mixture of oligosaccharides containing N-acetyl-D-glucosamine, D-mannose, and D-glucose varying in proportions from approximately 1/2.5/0.5 to 1/5/1.5 was obtained with glucosamine at the reducing end. Acid treatment of the lipid-bound oligosaccharide fraction yielded dolichyl pyrophosphate, suggesting that at least some of the oligosaccharides were linked to dolichol through a pyrophosphate group.
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PMID:Mannosyltransferase activity in calf pancreas microsomes. Formation of 14C-labeled lipid-linked oligosaccharides from GDP-D-[14C]mannose and pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate. 1 65

The aims of this study were to determine the effect of levels of various substances and reaction by-products, which are formed during hydrolysis of nucleic acids, on the derivatization and chromatography of nucleosides; and to investigate the silylation of mono- and dinucleotides. The effect of NaCl, KCl, MgCl2, NH4Cl, and (NH4)2SO4 on silylation and chromatography of nucleosides was studies at various molar excesses of salt. The response values for all nucleosides were studied at various molar excesses of salt. The response values for all nucleosides were significantly affected at molar excess salt present values (MSP) between 1 and 10 for KCl, NaCl, NH4Cl, (NH4)2SO4 and between 0.1 and 1 for MgCl2. It was noted that thymidine was more sensitive than other nucleosides if silylated in presence of these salts. Two chromatographic peaks at retention temperatures (RT) 240 and 251 were obtained for cytidine at MSP values of 10(-3) for NaCl, KCl, and MgCl2, and 10(-4) for NH4Cl and (NH4)2SO4. In a mixture of nucleosides the RT = 251 peak was used for quantitative analysis of cytidine as the RT = 240 peak elutes with guanosine. Thus, these salts have a significant effect on the gas-liquid chromatography of trimethylsilyl (TMS) cytidine in a mixture of nucleosides, especially the RT = 241 peak. The effect of salts on derivatization can be explained in part as follows: (a) reduced derivatization of nucleosides due to a decreased solubility in the solvent system; (b) formation of TMS anion derivatives, e.g. TMS-SO4, TMS-PO4, with a reduced molar excess of BSTFA; (c) metal chelation by Mg ions or other divalent cations with nucleosides or BSTFA; and/or (d) an increased breakdown of TMS derivatives in presence of salt in the sample or on the top 3 in. of the column packing. Also, experiments were made on the effect of other substances such as Tris, phosphate, alkaline phosphatase, and KCl on completeness of silylation. The individual impurities showed no significant effect on the relative weight response (RWR) values of nucleosides; however, when a mixture was used, significantly lower RWR values were observed for all nucleosides except thymidine when using 1000 molar excess of BSTFA greater than 1000 should be used for silylation and chromatography of nucleosides in an RNA hydrolysate. As reported earlier the best derivatization of nucleosides was achieved using closed tube silylation at 150 degrees for 15 min with 225 molar excess BSTFA and chromatography on 4% OV-11 on Supelcoport. In general, the presence of salts and other substances can be significant in quantitative work, thus it is suggested that they be removed using chromatographic cleanup methods. The stability of nucleosides as a function of concentration of HCl, at room temperature was studied and very low RWR values for nucleosides were obtained when stored for 48 h in greater than 0.001 N HCl. Trimethylsilylation of various nucleotides and dinucleotides were made at 15 min as a function of temperature, and at 150 degrees at different times...
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PMID:Derivatization and chromatography of nucleosides and nucleotides. 1 23

The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.
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PMID:The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein. 1 96

1. Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) in guinea pig thymus was extracted optimally in 10 mM Tris - HCl buffer at pH 8.0 containing 5 g/l Triton X-100. 2. alpha-Glycerophosphate, beta-glycerophosphate and phenolphthalein monophosphate were hydrolyzed by thymus extract with a pH optimum at 9.8-10.0, whereas p-nitrophenylphosphate and alpha-naphthylphosphate were hydrolyzed with pH optima at 10.7-10.8 and beta-naphthylphosphate at pH 11.2. P-Nitrophenylphosphate and phenolphthalein monophosphate proved to be the most suitable substrates. 3. Alkaline phosphatase was effectively inhibited by EDTA, Zn2+, histidine and urea therefore resembling the inhibition characteristics of alkaline phosphatase in the placenta and kidney, but not that in the liver and intestine, which differed markedly. 4. DEAE-cellulose chromatography and polyacrylamide disc electrophoresis revealed three enzyme peaks which did not differ in their substrate specificities and modifier characteristics. 5. Polyacrylamide disc electrophoresis of thymus, serum, placenta, kidney, liver, bone and intestine revealed no alkaline phosphatase bands definitely unique to thymus.
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PMID:Biochemical characterization of alkaline phosphatase in guinea pig thymus. 1 86

Portions of closed jejunal biopsies from the dog were homogenised and their organelles separated by isopycnic centrifugation on continuous sucrose density gradients. The distributions of marker enzymes for the principal organelles were determined using highly sensitive assay procedures. The following organelles, with assayed marker enzymes and modal densities between brackets were characterised: peroxisomes (catalase, 1.21); brush borders (zinc-resistant alpha-glucosidase, leucyl-beta-naphthyl-amidase, gamma-glutamyl transferase, alkaline phosphatase, 1.20); lysosomes (N-acetyl-beta-glucosaminidase, alpha-mannosidase, 1.19); mitochondria (malate dehydrogenase, 1.18); endoplasmic reticulum (Tris-resistant alpha-glucosidase, 1.16); basal-lateral membranes (5'-nucleotidase, 1.11) and cytosol (lactate dehydrogenase). Homogenisation in isotonic sucrose containing digitonin (0.12 mmol/litre) selectively disrupted lysosomes and increased the equilibrium density of brush border and basal-lateral membranes. This procedure will be used to study the subcellular pathology of naturally occurring intestinal disease in the dog.
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PMID:Subcellular fractionation studies on peroral jejunal biopsies from the dog. 3 Jan 25

Whole cells of Pseudomonas aeruginosa possess rhodanese activity. The enzyme can be released by rapidly resuspending the cells in cold Tris--HCl buffer. Approximately 95% of the rhodanese activity is released by cold shock. Release of the enzyme can be inhibited either by preincubating the cells with Mg2+ or by incorporating Mg2+ into the shocking buffer. The effect of Mg2+ can be reversed by washing the cells twice with buffer prior to cold shock. While rhodanese can be released from P. aeruginosa by cold shock, lactic dehydrogenase, a cytoplasmic enzyme, remains within the cell. Diazo-7-amino-1,3-napthalenedisulfonic acid, a compound which does not penetrate the cytoplasmic membrane, completely inactivated rhodanese and alkaline phosphatase, a periplasmic enzyme, whereas lactic dehydrogenase retained its full activity. These data suggest that rhodanese in P. aeruginosa, like alkaline phosphatase, is located distal to the cytoplasmic membrane in the periplasmic space. Electron micrographs also show that portions of the lipopolysaccharide outer membrane are shed from the cell during cold shock, while cells preincubated with Mg2+ did not release segments of their outer membrane.
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PMID:Release of rhodanese from Pseudomonas aeruginosa by cold shock and its localization within the cell. 11 Apr 32

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is reversibly inhibited by a variety of phenylarsonic acids, including some N-haloacetylated derivatives. The inhibition is of the competitive type, and Ki values are reported. The action on the enzyme of one of the arsonate inhibitors, the azo dye, 4-(4-aminophenylazo)-phenylarsonic acid was studied in detail, using spectrophotometric and kinetic methods. The azo dye binds more strongly to E. coli alkaline phosphatase than do the other arsonates. Spectrophotometric titration indicates the presence of a single, strong dye-binding site on the enzyme dimer molecule in the concentration range covered. In 0.1 M Tris - HCl buffer pH 8.0, 25 degrees C K diss for the dye - enzyme complex is 1.50 - 10(-5) M as determined by spectrophotometric titration. This value is in good agreement with the Ki = 1.30 - 10(-5) M obtained from kinetic measurements. The dye can be displaced from alkaline phosphatase by phosphate and competitive inhibitor 2-aminoethyl phosphonate. These results indicate that the dye binds with its arsonic acid group to the anion binding site of the active site of the enzyme. The binding of the dye to the native enzyme is associated with a red shift in the visible spectrum of the dye. It seems that the aromatic portion of the dye interacts with a hydrophobic region close to the anion binding site. The spectrum of the dye is not changed in the presence of the apoenzyme. When zinc is added to an apoenzyme-dye solution, the spectral changes of the dye depend on both the ratio of zinc per apoenzyme and the pH. The presence of Mg2+ had no effect on the observed phenomenon.
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PMID:Spectral studies of the interactions of Escherichia coli alkaline phosphatase with 4-(4-aminophenylazo)-phenylarsonic acid. 31 70

1. Specimens of human duodenal mucosa were obtained at duodenotomy. Superficial mucosal scrapings were homogenized in isotonic sucrose solution and fractionated by differential centrifugation. The distribution of organelles among the subcellular fractions was monitored by assay of suitable marker enzymes. 2. Enterokinase was recovered predominantly in the nuclear+brush-border fraction and 80% of the total activity was found to be particulate; approximately 20% of the enzyme was present in the soluble fraction, compared with 1% of the brush-border markers sucrase and alkaline phosphatase. 3. The brush-border-containing fraction was subfractionated by treatment with hypertonic Tris followed by differential and density gradient centrifugation. Enterokinase was distributed among the subfractions in parallel with brush-border markers and was concentrated in a subfraction which was highly enriched in microvillous membranes. 4. It was concluded that enterokinase is localized primarily to the microvillous membrane of the epithelial cell brush border in man, but that in addition a proportion of the enzyme may be present in a soluble or easily released form in the duodenal mucosa.
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PMID:Subcellular localization of enterokinase in human duodenal mucosa. 58 40


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