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)

The effect of tris(hydroxymethyl)aminomethane (Tris) buffer on outer membrane permeability was examined in a smooth strain (D280) and in a heptose-deficient lipopolysaccharide strain (F515) of Escherichia coli O8. Tris buffer (pH 8.00) was found to increase outer membrane permeability on the basis of an increased Vo of whole-cell alkaline phosphatase activity and on the basis of sensitivity to lysozyme and altered localization pattern of alkaline phosphatase. The Tris buffer-mediated increase in outer membrane permeability was found to be dependent upon the extent of exposure to and concentration of the Tris buffer. The Tris buffer effects were demonstrated not to be due to allosteric activation of cell-associated alkaline phosphatase and were specific for Tris buffer. Exposure of cells to Tris resulted in the release of a limited amount of cell envelope component. Investigators utilizing Tris buffer are cautioned that Tris is not physiologically inert and that it may interact with the system under investigation.
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PMID:Tris(hydroxymethyl)aminomethane buffer modification of Escherichia coli outer membrane permeability. 700 85

The barrier function of the Escherichia coli outer membrane against low concentrations of maltose in strains missing the lambda receptor was partially overcome by treating the cells for 3 h with 25 mM Ca2+. Kinetic analysis of maltose-transport revealed a Ca2+-induced shift of the apparent Km of the system from about 100 microM in cells pretreated with Tris to about 15 microM in cells pretreated with Tris plus Ca2+. In contrast to maltose transport in untreated cells, that of Ca2+-treated lamB cells was inhibited by molecules with a high molecular weight, such as amylopectin (molecular weight, 20,000), and anti-maltose-binding protein antibodies. In addition, lysozyme was shown to attack Ca2+-treated cells in contrast to untreated cells. The Ca2+-induced permeability increase of the outer membrane allowed reconstitution of maltose transport in a mutant missing the maltose-binding protein with osmotic shock fluid containing the maltose-binding protein. Even though Ca2+-treatment allowed the entry of large molecules, the release of the periplasmic maltose-binding protein or alkaline phosphatase was negligible.
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PMID:Reconstitution of maltose transport in malB mutants of Escherichia coli through calcium-induced disruptions of the outer membrane. 701 12

1. A study has been carried out on the steady-state kinetics followed by the alkaline phosphatase from Escherichia coli at different pH, temperatures, ionic strengths, phosphate concentrations and in the presence of the effectors such as Tris, NH4+--NH3 and CH3OH; p-nitrophenyl phosphate was used as substrate. 2. Contrary to what has generally been accepted, in most cases the enzyme follows non-Michaelian kinetics for a wide substrate concentration range, giving concave-down Lineweaver-Burk plots. Only at high phosphate concentrations (5 . 10(-3) M) and at high ionic strengths (2.0 M) is a linear Lineweaver-Burk plot obtained (Michaelian kinetics). 3. In order to analyse the kind of kinetics obtained, a non-linear regression fitting method was used to obtain rate vs substrate concentration equations as polynomial quotients of minimum degree with positive coefficients. 4. Most of the data obtained follows 2:2 degree type equations. 5. These results tend to suggest an idea of cooperativity rather than one of independence between the sites of the dimeric enzyme. A model is discussed for cooperativity between the sites with a wide concentration range giving concave-down Lineweaver-Burk plots.
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PMID:Negative cooperativity in alkaline phosphatase from E. col: new kinetic evidence from a steady-state study. 704 Jan 34

Thermal damage to the outer membrane of Escherichia coli W3110 was studied. When E. coli cells were heated at 55 degrees C in 50 mM Tris-hydrochloride buffer at pH 8.0, surface blebs were formed on the cell envelope, mainly at the septa of dividing cells. Membrane lipids were released from the cells during the heating period, and part of the released lipids formed vesicle-like structures from the membrane. This vesicle fraction had a lipopolysaccharide to phospholipid ratio similar to that of the outer membrane of intact cells, whereas it had a lower content of protein than the isolated outer membrane. After heating bacterial cells at 55 degrees C for 30 min, the resulting leakage from the cells of a periplasmic enzyme, alkaline phosphatase, amounted to 52% of the total activity, whereas no release of a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase, was detected. The results obtained suggest that surface blebs formed by heat treatment almost completely consist of the outer membrane and that the blebs may be gradually released from the cell surface into the heating menstruum to partially form vesicles.
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PMID:Heat-induced blebbing and vesiculation of the outer membrane of Escherichia coli. 705 91

1. Hen's egg yolk alkaline phosphatase can transfer the donor substrate phosphoryl to a hydroxyl containing acceptor. 2. This acceptor contains preferably an alpha-amino or alpha-imino group. 3. The optimal pH of the transfer reaction is at 9.45 with Tris and at 9.60 with diethanolamine as acceptor. 4. Transfer products were isolated and characterized. 5. The ratio of products is independent of the donor substrate. ROH and R'OP production increase linearly with the acceptor concentration, while Pi release and Km remain constant. 6. This points to a mechanism involving two phosphorylated intermediates. 7. The acceptor bypasses the isomerization step of the phosphoryl enzyme, which is necessary before hydrolysis can take place.
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PMID:Transphosphorylation mechanism of hen's egg yolk alkaline phosphatase. 710 52

A simple and rapid microenzyme-linked immunosorbent assay has been developed for determination of anti-poly(ADP-ribose) antibodies in humans using a combination of protein A-alkaline phosphatase conjugates and poly(ADP-ribose)-coated polyvinyl microplates. After a 1-h treatment of the plates with 100 microliters of poly L-lysine (PLL) solution (50 micrograms/ml), an aliquot of the solution containing 100 ng poly(ADP-ribose) (50 microliters) was added to the PLL-treated plates and evaporated at 37 degrees C overnight to facilitate the adherence of poly(ADP-ribose) to the plates. Nonspecific binding of diluted test sera from patients with systemic lupus erythematosus (SLE) or from normal individuals to the PLL-coated plates was minimized by exposure of the plates for 1 h to Tris-buffered saline (pH 7.4) containing 0.01% bovine serum albumin (BSA). This method was also applicable to the determination of anti-double-stranded DNA antibodies in humans. The present assay is advantageous over those reported so far as it saves time and antigen.
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PMID:A simple and rapid microenzyme-linked immunosorbent assay for antibodies to poly(ADP-ribose) in systemic lupus erythematosus. 714 14

Alkaline phosphatase activity was found in human thyroid homogenate at a specific activity of about 0.01 units/mg protein. The enzyme responsible appears to be a membrane-bound sialoglycoprotein. The properties of thyroid membrane alkaline phosphatase were examined after extraction with non-ionic detergent or butanol. It was detected mainly as a single electrophoretic form with a mobility similar to that of the liver form of the enzyme. It was readily inhibited by homoarginine, but not by phenylalanine. It reacted in Ouchterlony double diffusion with antiserum raised against liver alkaline phosphatase, but not with antiserum to placental alkaline phosphatase. Heat treatment at 56 degree C for 10 min at pH 7.5 resulted in an approx. 50% loss of enzyme activity. Its relative molecular mass was estimated to be 320000 by gel filtration, and 300000 by gradient gel electrophoresis. It required magnesium for full activity, and had a pH optimum of 10.5 in Tris-borate buffers. It was concluded that thyroid alkaline phosphatase belongs to the liver/bone/kidney isoenzyme group, but may exist in a molecular form distinct from others previously described.
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PMID:Alkaline phosphatase from human thyroid. 731 84

To investigate the variability in test results obtained with the SOS chromotest (Escherichia coli PQ37 genotoxicity assay) when varying the composition of the exogenous metabolizing system (S9 mix), we examined the influence of different S9 and NADP concentrations, of buffer pH value, of SDS concentrations, the effects of E. coli PQ37 density and centrifugation steps on the expression of beta-galactosidase (beta g) and alkaline phosphatase (ap) activity, the calculated induction factors (IFs) and SOS-inducing potencies (SOSIPs). Additionally we examined the metabolic potency (stability) of S9 mix when stored at 37 degrees C before use. Initially, we used 0-5000 ng (= 0-20 nmole) benzo[a]pyrene (B[a]P) as a reference compound for the test procedure in the presence of standard S9 mix. Subsequently, to evaluate the results of S9 mix variations we examined several polycyclic aromatic hydrocarbons (PAHs) using both the standard and a modified S9 mix composition and test protocol. We observed the highest beta g and ap activities and/or IFs using only 11-27 microliters 9000 x g liver supernatant (S9) from Aroclor 1254-induced rats per assay (20-50% of standard amount) and calibrating the S9 mix Tris buffer to pH 7.8-8.0. 60-300 micrograms NADP/assay (10-50% of standard) was sufficient for optimum activation of PAHs. In contrast to previous investigations about the variability of the SOS chromotest in the absence of a metabolizing system, higher induction factors were obtained when using higher bacterial densities (12-18 x 10(6) cfu/assay). Centrifugation steps as recommended by other investigators were not necessary when using optimum S9 amounts. The metabolic activity of S9 mix remained nearly constant approximately 20 min after preparation, but decreased to 80% of its activity in about 1 h.
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PMID:Influence of S9 mix composition on the SOS response in Escherichia coli PQ37 by polycyclic aromatic hydrocarbons. 767 15

The hydrolysis and transphosphorylation reactions of a series of phosphate monoesters, ROPO3(2)-(R = 2,4-dinitrophenyl, 4-nitrophenyl, phenyl, glucose-1, glycerol-1, methyl, ethyl, and dodecyl), catalyzed by Escherichia coli alkaline phosphatase and a mutant enzyme, Ser102Cys, have been studied at alkaline pH using the rates of change in the 31P NMR signals of substrate, the hydrolysis product (inorganic phosphate), and the transphosphorylation product (O-Tris phosphate) as the assay. The kcat at pH 8.0 for the wild-type enzyme is approximately 30 s-1 and is independent of the nature of the R group, when the pKa of the leaving group is < 10. Under these conditions the rate of phosphorylation is much faster than dissociation of inorganic phosphate, 15-60 s-1. If the pKa of the leaving group is between 10 and 15, phosphorylation and dissociation of the product phosphate both contribute to the rate limit. If the pKa of the leaving group is > 15, phosphorylation is rate limiting. A Bronsted plot of log kcat vs pKa of the leaving group for those substrates for which phosphorylation is rate limiting yields a beta lg of approximately -0.6. In contrast to the wild-type enzyme, the log kcat values for the S102C mutant enzyme catalyzing the hydrolysis of phosphate esters are linearly dependent on the pKa's of the leaving group throughout the range of pKa from 4 to 16. Phosphorylation of C102 is the rate controlling step, and kcat is independent of the Tris concentration as predicted for rate limiting phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dependence of the phosphorylation of alkaline phosphatase by phosphate monoesters on the pKa of the leaving group. 770 37

His-412 in wild-type Escherichia coli alkaline phosphatase is a direct ligand to one of the two zinc atoms critical for the function of the enzyme. To investigate the function of this residue, site-specific mutagenesis was used to substitute His-412 with asparagine and alanine, generating mutant enzymes H412N and H412A, respectively. Both mutant enzymes show a 5-fold decrease in kcat and 30-fold increase in Km when compared to the corresponding kinetic parameters for the wild-type enzyme. In contrast to the wild-type enzyme, Tris and ethanolamine inhibit both the mutant enzymes by inhibiting the hydrolysis reaction and not participating in the transferase reaction; furthermore, both mutants have lower zinc and phosphate content than the wild-type enzyme. The addition of Zn2+ to the H412N and H412A enzymes restores catalytic activity to within 2-fold of the value for the wild-type enzyme, but more importantly the presence of Zn2+ completely restores substrate affinity. The similarity in the kinetic parameters for the H412N and H412A enzymes in the absence and presence of zinc suggests that the asparagine side chain does not play a significant role in coordinating zinc. Furthermore, both the asparagine and alanine substitutions reduce the affinity of the resulting enzymes for zinc. The pH profiles for the two mutant enzymes are different than the pH profile observed for the wild-type enzyme, suggesting that the amino acid substitutions may have altered the pKa of the zinc coordinated water molecule that is critical in the second step of the mechanism. These data suggest that His-412 does not directly participate in the catalytic mechanism but is mainly involved in zinc binding, and therefore is also indirectly involved in substrate binding and product release.
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PMID:Mutations at histidine 412 alter zinc binding and eliminate transferase activity in Escherichia coli alkaline phosphatase. 798 32


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