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 method has been developed for the detection of Brucella abortus in complex tissue homogenates. The technique uses tissue homogenization in the presence of sucrose and Triton X-100 and subsequent filtration through a 5-microns pore size filter to remove mammalian nuclei and cellular debris. The DNA from the bacteria is then extracted, dot blotted onto nitrocellulose, and hybridized with a biotinylated probe of B abortus strain 19 DNA. In the present study, BALB/C mice were inoculated intraperitoneally with either 10(9) or 10(11) B abortus strain 2308S organisms. After 6 days, the mice were euthanatized by cervical dislocation and the livers were removed, weighed, and the appearance of each was noted. The tissues were homogenized, and a viable cell count was performed to determine the number of bacteria in each organ. The DNA was extracted, blotted onto nitrocellulose, and hybridized with the Brucella probe. The biotin label was detected by use of a commercially available streptavidin/alkaline phosphatase system. In control experiments, the technique detected 10(5) organisms in a mixture of bacteria and 1 g of rat liver. The technique also detected 10(7) B abortus organisms/g of tissue from experimentally inoculated mice. The probe was specific for Brucella and had no affinity for contaminating bovine or bacterial DNA.
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PMID:Detection of Brucella abortus in mammalian tissue, using biotinylated, whole genomic DNA as a molecular probe. 251 17

Intramembranous localization of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3, 1, 3, 1; AlPase) was observed biochemically in Bacillus megaterium KM grown in 1% polypeptone medium containing 0.5% NaCl at 37 degrees C under aerobic conditions and harvested at the latter logarithmic phase. AlPases from B. megaterium have been separated into soluble and membrane-bound forms by the centrifugation after cell disruption by sonication. The membrane-bound enzyme was further fractionated to two forms by phase separation using a non-ionic detergent, Triton X-114; one was successfully solubilized into the aqueous phase and the other remained in the Triton phase. Both AlPases of sonication- and Triton-solubilized forms were partially purified by gel filtration and anion-exchange column chromatographies. Their molecular weights were different (52,000 for soluble and 66,000 for Triton-solubilized forms) and the Vmax of the sonication-solubilized enzyme (227 nmol/min/mg protein) was 11-fold higher than that of the Triton-solubilized one although similar Km values (1.7 and 2.3 mM) were observed. Optimum pH of these enzymes tended to shift to a neutral range during the purification steps. These results suggest the multiplicity of AlPase anchoring to the membranes; 1) sonication-solubilized form which may be buried within the membrane lipids by its hydrophobic peptide and solubilized by the cell disruption, 2) detergent-solubilized form which may be bound loosely to the membrane by its hydrophobic domain and solubilized due to the amphiphilicity of enzyme protein, and 3) insolubilized form which may be bound fast to the membrane by its strong hydrophobicity and also have the function of enzymatic ability.
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PMID:[Biochemical studies on intramembranous localization of alkaline phosphatase in Bacillus megaterium KM]. 251 45

In the presence of inhibitors for mitochondrial H+-ATPase, (Na+ + K+)- and Ca2+-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane ("ecto-ATPases"). These ATPases accept several nucleotides, are stimulated by Ca2+ and Mg2+, and are inhibited by N.N'-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brush-border and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg2+ and Mn2+, but not by Ca2+, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator. ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H+ secretion is electrogenic and requires either exit of a permeant anion (Cl-) or entry of a cation, e.g., Na+ via electrogenic Na+/D-glucose and Na+/L-phenylalanine uptake. In the presence of Na+, ATP-driven H+ efflux is stimulated by blocking the Na+/H+ exchanger with amiloride. These data prove the coexistence of Na+-coupled substrate transporters, Na+/H+ exchanger, and an ATP-driven H+ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H+ pumps with (a part of) the DCCD- and NEM- sensitive ATPases at the cytosolic side of the brush-border membrane.
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PMID:Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion. 253

A fraction of intestinal alkaline phosphatase (IAP) is secreted into blood. To study this process, enzyme secretion was examined in a fetal (IRD-98) and a differentiated (Caco-2) intestinal cell line. Tissue-unspecific alkaline phosphatase (AP) activity in the IRD-98 cells increased 20-fold after addition of 1.5 mM sodium butyrate and 40 mM NaCl, but no AP activity was secreted into the medium. In contrast, newly synthesized IAP in Caco-2 cells was secreted into the medium. AP secretion increased with time and was inhibited by monensin. Medium AP was still partially bound to membranes as assessed by Triton X-114 phase separation and could be released by the addition of serum. Analysis by sodium dodecyl sulfate polyacrylamide gels and by isoelectric focussing showed that secreted AP gave a pattern similar to that of the AP released from membranes by phospholipase D treatment. When Caco-2 cells were grown on filters, AP activity was found in both basolateral (75%) and luminal (25%) media. These data demonstrate that the secretion of a particulate AP with extracellular release from the membrane can account for the appearance of the intestinal isozyme in both the serum and the lumen.
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PMID:Intestinal alkaline phosphatase is secreted bidirectionally from villous enterocytes. 254 40

Alkaline phosphatase from calvaria of 8 to 12-day-old Wistar rats was purified to electrophoretic homogeneity by a simple procedure (homogenisation, solubilisation by Triton X-100, DEAE-Sephacel ion exchange chromatography). For the holoenzyme, a Mr of about 160 kDa was determined, and it seems to consist of two identical subunits. The pH optima for the hydrolysis of phosphoethanolamine and p-nitrophenylphosphate are 10.0 and pH 9.0-10.5, respectively. The rate constants for the hydrolysis of phosphoethanolamine, p-nitrophenylphosphate and other phosphomonoesters at pH 10.0 are comparable, but the Km values differ by one to two orders of magnitude. At physiological pH (7.5) the maximum hydrolysis rate of the substrates phosphoethanolamine and p-nitrophenylphosphate was only 8% and 5%, respectively, of that determined at the pH optimum. On the basis of the kinetic data an in vivo function of alkaline phosphatase in bones as a monophosphate ester hydrolyzing enzyme seems unlikely.
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PMID:[Phosphoethanolamine--a substrate of alkaline phosphatase isolated from rat calvaria]. 261 22

The interaction of alpha-chymotrypsin, invertase, alcohol dehydrogenase and alkaline phosphatase with some ionic and non-ionic surfactants, viz. sodium dodecyl sulphate, dioctyl sodium sulphosuccinate, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide and Triton X-100, has been examined by studying the effect of varying surfactant concentrations on enzyme activities as well as by determining the time-dependent inactivation and the time-independent inhibition. The kinetic parameters, Km and Vmax, for alpha-chymotrypsin-catalysed reaction in presence of sodium dodecyl sulphate were evaluated. Anionic surfactants markedly decreased enzyme activity, whereas cationic surfactants were less effective. Nonionics showed no effect. This change in enzyme activity was also dependent on the nature of enzyme.
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PMID:Stability and kinetic behaviour of some enzymes in surfactant environment. 263 63

The activities of lysosomal maltase in the serum, bile and liver were determined in intrahepatic cholestasis rats induced by alpha-naphtylisothiocyanate (ANIT, 200 mg/kg, i.p.), and compared with changes in alkaline phosphatase (ALP) activity. Moreover, the influences of endogenous bile acids on the release of maltase activity from the liver in intrahepatic cholestasis rats were studied. The maltase activities in the serum and bile significantly increased from 4 and 8 h after the intraperitoneal administration of ANIT, respectively. Conversely, a significant decrease in liver maltase activity was observed from 4 h after the injection of ANIT. On the other hand, total bile acid concentrations in the serum and bile significantly increased immediately after the treatment of ANIT, when biliary bile acid, exogenous bile acid or Triton X-100 was added to lysosomal fraction in the liver, the maltase activity in the supernate after the reaction significantly increased in proportion to the concentration of each substance added to the liver lysosome. These results suggested that maltase might be released from liver lysosomal membrane by surface active-action of bile acid accumulated in the liver after the administration of ANIT. Moreover, the changes in ALP activities in the serum, bile and liver after the administration of ANIT were almost similar to those in maltase activity.
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PMID:[The influences of endogenous bile acids on maltase and alkaline phosphatase activities in intrahepatic cholestasis rats]. 269 43

Mouse embryos were extracted with 0.5% Triton X-100 and subjected to cellulose acetate electrophoresis. In fertilized eggs, two forms of alkaline phosphatase (ALP), a slow-moving form and a fast-moving form, were observed. As cleavage proceeded, the fast-moving form disappeared, and the slow-moving form, the mobility of which was similar to that of the slow-moving form of the kidney, became gradually dominant up to the blastocyst stage (named 'embryonic' form). With blastulation, another fast-moving form showing a similar mobility to the lung ALP began to appear in blastocysts and showed a transient dominance in hatched blastocysts. After implantation, both the embryonic form and the fast-moving form gradually faded, and were eventually replaced by the new form, which may be named 'fetal form' in Day 7 embryos. These results clearly demonstrated that ALP activity does exist in embryos at all stages of preimplantation development. Moreover, the changes in multiple forms of ALP correlated with embryonic development may suggest that these multiple forms may have differential roles in the process of early development.
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PMID:Multiple forms of alkaline phosphatase in mouse preimplantation embryos. 275 58

The larval midgut epithelial cell of the silkworm, Bombyx mori, has two forms of alkaline phosphatase and trehalase, soluble and membrane-bound. Alkaline phosphatase and trehalase of the latter form are found in the brush border membrane and the basolateral membrane, respectively. In this work we studied the membrane anchors of these membrane-bound enzymes. Alkaline phosphatase was solubilized by phosphatidyl-inositol-specific phospholipase C, but not by papain. Conversely, trehalase was released from the membrane by papain, but not by phosphatidylinositol-specific phospholipase C. Both enzymes were solubilized in an amphiphilic form with 0.5% Triton X-100 plus 0.5% sodium deoxycholate (pH 7.0). The detergent-solubilized alkaline phosphatase and trehalase were converted to hydrophilic form on incubation with phosphatidylinositol-specific phospholipase C and papain, respectively. The effects of papain on solubilization and conversion of trehalase were completely inhibited by leupeptin. These results suggest that, in the silkworm larvae, alkaline phosphatase is anchored in the brush-border membrane via a glycosyl-phosphatidylinositol, while trehalase is associated with the basolateral membrane through a hydrophobic segment of the polypeptide.
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PMID:Membrane anchors of alkaline phosphatase and trehalase associated with the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori. 276 26

Renal dipeptidase (dehydropeptidase-I, EC 3.4.13.11) was released from pig kidney membrane preparations by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus and Bacillus thuringiensis and a phospholipase C preparation from Bacillus cereus to a similar extent as alkaline phosphatase. Endopeptidase-24.11 and aminopeptidase N were not released by this treatment. After treatment of the membrane fraction with the S. aureus phospholipase C the dipeptidase was converted from an amphipathic to a hydrophilic form, as deduced from phase-separation experiments in Triton X-114. It is concluded that renal dipeptidase is anchored to the microvillar membrane by covalently attached phosphatidylinositol.
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PMID:Renal dipeptidase is one of the membrane proteins released by phosphatidylinositol-specific phospholipase C. 282 7


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