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 nonisotopic enzymolysis assay method of phosphatidylinositol kinase (PIK) has been developed. Phosphatidylinositol 4-phosphate (PIP), phosphorylated from phosphatidylinositol (PI) by PIK was hydrolyzed with phosphainositide-specific phospholipase C. The product, inositol 1, 4-bisphosphate (IP2), was separated from inositol 1-phosphate (IP) with Dowex-1 column chromatography. Then, the IP2 was hydrolyzed by alkaline phosphatase to yield PI, and the PI was determined by colorimetry. The PIK activity was defined as PI nmol/mg protein per min. The recovery was 91%, CV = 6.5%
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PMID:[A nonisotopic enzymolysis assay method of phosphatidylinositol kinase]. 166 83

1. Phosphatidylinositol 4-phosphate (PtdIns4P) is degraded by isolated membranes from Xenopus laevis oocytes. 2. Incubation of [4-32P]PtdIns4P with membranes yields only radioactive inorganic phosphate, indicating the presence of a phosphomonoesterase. 3. Membranes hydrolyze Ptd[2-3H]Ins4P to produce mainly Ptd[2-3H]Ins in the lipid phase. In this incubation [3H]inositol and inositol monophosphate appear in the water phase. 4. Membrane incubations of Ptd[2-3H]Ins4P carried out in the presence of excess non-radioactive Ins(1,4)P2 allows the trapping of small amounts of [3H]Ins(1,4)P2. These results demonstrate the presence of a phospholipase C. 5. Testing several phosphorylated analogs, it is determined that fructose 1,6-bisphosphate and alpha-glycerophosphate are potent inhibitors of the oocyte PtdIns4P phosphomonoesterase.
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PMID:The hydrolysis of phosphatidylinositol 4-phosphate in membranes of Xenopus laevis oocytes: characteristics of a phosphomonoesterase. 166 8

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

In Pseudomonas aeruginosa, the genes pilB, pilC, and pilD encode proteins necessary for posttranslational modification and assembly of pilin monomers into pilus organelles (D. Nunn, S. Bergman, and S. Lory, J. Bacteriol. 172:2911-2919, 1990). We show that PilD, encoding a putative pilin-specific leader peptidase, also controls export of alkaline phosphatase, phospholipase C, elastase, and exotoxin A. pilD mutants accumulate these proteins in the periplasmic space, while secretion of periplasmic and outer membrane proteins appears to be normal. The periplasmic form of exotoxin A was fully mature in size, contained all cysteines in disulfide bonds, and was toxic in a tissue culture cytotoxicity assay, suggesting that in pilD mutants, exotoxin A was folded into its native conformation. The function of the other two accessory proteins, PilB and PilC, appears to be restricted to pilus biogenesis, and strains carrying mutations in their respective genes do not show an export defect. These studies show that in addition to cleaving the leader sequence from prepilin, PilD has an additional role in secretion of proteins that are released from P. aeruginosa into the surrounding media. PilD most likely functions as a protease that is involved in processing and assembly of one or more components of the membrane machinery necessary for the later stages of protein extracellular localization.
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PMID:Multiple roles of the pilus biogenesis protein pilD: involvement of pilD in excretion of enzymes from Pseudomonas aeruginosa. 167 84

Surface charge of Leishmania mexicana amazonensis was investigated by direct zeta-potential determination and ultrastructural cytochemistry, and its surface tension was studied by measurements of the advancing contact angle formed by the parasite monolayers with drops of liquids of different polarities. Both virulent and avirulent promastigotes exhibited negatively charged surfaces with a zeta-potential of about -15 mV. Treatment of these cells with trypsin, alkaline phosphatase, or phospholipase C rendered their surfaces less negatively charged, whereas neuraminidase did not alter the parasite negativeness. Cytochemically, we could observe a reduction in the cationized ferritin binding after the parasite treatment with each of the former enzymes, but not with neuraminidase. The surface free energy of parasites was calculated by taken to account the London dispersion, the Keeson dipole-dipole, and the Debye dipole-induced forces, as well as the surface polarity of the parasites and their zeta-potentials, by considering their adhesion to polystyrene surfaces. The delta G values of -6.4 and -18.1 mJ.m-2 were obtained for avirulent and virulent promstigotes, respectively.
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PMID:The surface free energy of Leishmania mexicana amazonensis. 170 80

The number of identifiable stages and expression of differentiation markers in cells of the osteoblast lineage are not well understood. In the present study, a mAb, designated rat bone marrow (RBM) 211.13, was prepared that stained selectively the osteogenic and preosteoblastic cells along the surfaces of bone in calvariae, femurs, and metatarsals. The staining was cell surface associated and coincided with that for alkaline phosphatase (APase) detected histochemically. Only cells positive for APase activity by biochemical assay and not those without APase activity (e.g., fetal rat skin) stained with RBM 211.13. By immunoblotting, RBM 211.13 recognized a band coinciding with APase activity on nonreducing/nondenaturing gels, and RBM 211.13 precipitated a protein which on reduced gels migrated with an apparent molecular mass of approximately 80 kD. RBM 211.13 labeling was abolished by phosphatidylinosital-specific phospholipase C, known to release APase from the cell surface. All of these data support the concept that RBM 211.13 recognizes the bone isoenzyme of APase. RBM 211.13 was used to sort by flow cytometry the APase-positive and APase-negative cells from mixed fetal rat calvaria (RC) cell populations. The osteoprogenitors we identified earlier that form bone nodules in vitro (Bellows, C. G., J. E. Aubin, J. N. M. Heersche, and M. E. Antosz. 1986. Calcif. Tissue Int. 36:143-154; Bellows, C. J., J. N. M. Heersche, and J. E. Aubin. 1990. Dev. Biol. 140:132-138) were found within the APase-positive pool. By immunopanning, RC cells were separated into APase-enriched (APase-positive, adherent) and APase-depleted (APase-negative, nonadherent) populations. The APase-positive fraction was enriched two-to-threefold for bone-forming osteoprogenitors compared to unfractionated cells, while the APase-negative population formed very few nodules under the same conditions. Both populations responded to the glucocorticoid dexamethasone (DEX) with an increase in bone nodule formation. However, the fold stimulation in bone formation in the APase-negative population was approximately 30-fold, while the fold stimulation in the APase-positive population was only approximately 5-fold. These data suggest that APase expression can be used for immunoselection to fractionate osteoblastic populations into an APase-positive population and a population initially APase-negative, that virtually all osteoprogenitors forming bone in vitro in the absence of added glucocorticoids reside in the APase-positive pool, and that the only osteoprogenitors present in the APase-negative pool are those requiring DEX to differentiate.
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PMID:Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells. 171 92

The antigen detected by monoclonal antibodies reacting with human osteosarcoma-associated antigen was shown to be a phosphatidyl-inositol (PI)-glycan-anchored protein, which can be released from the cell surface by PI-specific phospholipase C-treatment. The antigen detected by 2D3 and 2H10 antibodies exhibited alkaline phosphatase activity. Both antibodies strongly reacted with bone-type alkaline phosphatase. However, importantly, immunohistochemical analysis demonstrated that 2D3 and 2H10 did not react with alkaline phosphatase present in kidney or liver. In addition, neither placental nor intestinal alkaline phosphatase was recognized by 2D3 and 2H10 antibodies. These results indicated that two monoclonal antibodies, 2D3 and 2H10, are highly specific for bone-type alkaline phosphatase and can distinguish bone alkaline phosphatase from liver alkaline phosphatase in spite of the fact that liver and bone alkaline phosphatase are encoded by the same gene.
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PMID:Detection of bone-type alkaline phosphatase by monoclonal antibodies reacting with human osteosarcoma-associated antigen. 171 40

We have isolated and characterized four toxin A excretion-deficient mutants of Pseudomonas aeruginosa PAO1. Similar to previously described mutants (B. Wretlind and O. R. Pavlovskis, J. Bacteriol. 158:801-808, 1984), the mutants appear to have a pleiotropic defect in the excretion of several extracellular products, including toxin A, elastase, alkaline phosphatase, and phospholipase C. However, the mutants are not defective in the excretion of either alkaline protease or exoenzyme S. We also examined the localization and processing of toxin A in these mutants by using pulse-labeling experiments. Mature toxin A was found to be localized to the membranes only. Our results suggest that toxin A is localized to the outer membrane but is not exposed to the extracellular surfaces of the outer membranes. The results also suggest that toxin A obtained from the excretion-deficient mutants has intact disulfide bonds.
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PMID:Isolation and characterization of toxin A excretion-deficient mutants of Pseudomonas aeruginosa PAO1. 173 Apr 83

Subcellular fractionation of pig kidney cortex revealed that aminoacylase I (EC 3.5.1.14, N-acyl-L-amino-acid aminohydrolase) is predominantly a soluble enzyme with only 0.5% of the total activity being recovered in the membrane fraction. The aminoacylase I activity associated with the membrane preparations displayed neither rapid release following incubation with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis nor the distinctive differential pattern of detergent solubilization which was seen with glycosyl-phosphatidylinositol-anchored proteins (renal dipeptidase, alkaline phosphatase). When fractionated by phase separation in Triton X-114, integral membrane proteins of kidney microvillar membranes partitioned predominantly (greater than 90%) into the detergent-rich phase. In contrast, only 3.7% of aminoacylase I activity associated with microvillar membranes partitioned into the detergent-rich phase. Aminoacylase I activity of pig kidney would therefore appear to be a hydrophilic protein in nature and is not, as suggested previously, a G-PI-anchored integral membrane protein.
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PMID:Aminoacylase I is not a glycolipid-anchored ectoenzyme in pig kidney. 182 88

Doxorubicin, when incubated for 30 minutes with [32P]-labelled human erythrocyte membrane vesicles, produced an elevation of [32P]inositol-1,4,5-trisphosphate levels. The maximum rise was obtained with 10(-8) mol/l doxorubicin [132 (S.E. 13%) of control, n = 6, P = 0.001]. However, when the inositol lipids were examined, there was no evidence that doxorubicin stimulated the breakdown of [32P]phosphatidylinositol-4,5-bisphosphate under resting conditions, suggesting that the elevated levels of [32P]inositol 1,4,5-trisphosphate were not the result of the stimulation of phospholipase C. Instead, it was found that the dephosphorylation of inositol 1,4,5-trisphosphate by a 5'-phosphomonoesterase was partially inhibited by 10(-8) mol/l doxorubicin so that the rise in [32P]inositol 1,4,5-trisphosphate resulted from the inhibition of the breakdown of constitutively released [32P] inositol 1,4,5-trisphosphate. Similar data was also obtained with another aminoglycoside antibiotic, neomycin. The release of [32P] inositol 1,4-bisphosphate and [32P] inositol 1,4,5-trisphosphate and the breakdown of the inositol lipids in response to calcium (2.5 x 10(-4) and 10(-3) mol/l) stimulation was enhanced by doxorubicin (10(-6) to 10(-12) mol/l). These effects on resting and stimulated inositol lipid metabolism are discussed with reference to the paradoxical effects of doxorubicin to both stimulate and inhibit proliferation, according to concentration.
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PMID:Doxorubicin interactions at the membrane: evidence for a biphasic modulation of inositol lipid metabolism. 183 96


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