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 function of aspartic acid residue 101 in the active site of Escherichia coli alkaline phosphatase was investigated by site-specific mutagenesis. A mutant version of alkaline phosphatase was constructed with alanine in place of aspartic acid at position 101. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, pH 8.0, both the kcat and the Km for the mutant enzyme increase by approximately 2-fold, resulting in almost no change in the kcat/Km ratio. Under conditions of no external phosphate acceptor and pH 8.0, both the kcat and the Km for the mutant enzyme decrease by approximately 2-fold, again resulting in almost no change in the kcat/Km ratio. The kcat for the hydrolysis of 4-methyl-umbelliferyl phosphate and p-nitrophenyl phosphate are nearly identical for both the wild-type and mutant enzymes, as is the Ki for inorganic phosphate. The replacement of aspartic acid 101 by alanine does have a significant effect on the activity of the enzyme as a function of pH, especially in the presence of a phosphate acceptor. At pH 9.4 the mutant enzyme exhibits 3-fold higher activity than the wild-type. The mutant enzyme also exhibits a substantial decrease in thermal stability: it is half inactivated by treatment at 49 degrees C for 15 min compared to 71 degrees C for the wild-type enzyme. The data reported here suggest that this amino acid substitution alters the rates of steps after the formation of the phospho-enzyme intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity. 268 45

The Dictyostelium discoideum alkaline phosphatase was investigated kinetically in an attempt to elucidate its mechanism of action. Analysis of the hydrolysis of p-nitrophenyl phosphate by stopped-flow spectrophotometry revealed biphasic kinetics, suggesting a double displacement enzyme mechanism. Furthermore, Tris stimulated activity in an uncompetitive manner, a result that was consistent with this interpretation. The enzyme was inhibited reversibly by phosphate at low ionic strength, but the inhibition was irreversible at high ionic strength and the latter effect was enhanced at alkaline pH values. These results indicate that high ionic strength and alkaline pH conditions bring about a conformational change that renders the enzyme susceptible to irreversible inhibition by phosphate.
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PMID:Studies on the mechanism of action of the alkaline phosphatase from Dictyostelium discoideum. 270 77

The C-terminal two-thirds of the rat liver ATP synthase beta subunit has been overexpressed and exported to the Escherichia coli periplasm under the direction of the alkaline phosphatase (phoA) promoter and leader peptide. The processed soluble protein contains the 358 amino acids from glutamate 122 to the rat liver beta C-terminal serine 479, including all the regions that have been predicted by chemical and genetic modification studies to be involved in nucleotide, Pi, and Mg2+ binding. Through a simple sequence of Tris/EDTA/lysozyme treatment, osmotic lysis, and alkaline pH washes, the processed beta subunit fragment can be prepared in greater than 95% purity and at a yield of greater than 20 mg/liter of culture. It interacts with 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) which exhibits a strong enhancement of fluorescence upon binding. A similar enhancement is observed upon interaction with TNP-ADP. Enhancement observed with both TNP-nucleotides is markedly reduced by prior addition of either ATP or ADP and almost completely prevented by the ATP synthase inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. Both TNP-ATP and TNP-ADP bind at a stoichiometry of approximately 1 mol of nucleotide/mol of beta subunit fragment. Under the same conditions, TNP-AMP does not exhibit a fluorescence enhancement. This work demonstrates that, in the absence of interaction with other ATP synthase subunits, the rat liver beta subunit sequence from glutamate 122 to the C terminus exhibits no more than one readily detectable nucleotide binding domain. This success in producing a "functional" beta subunit fragment has significance for the pursuit of genetic and physical studies focused on the structure and function of the rat liver ATP synthase beta subunit.
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PMID:Mitochondrial ATP synthase. Overexpression in Escherichia coli of a rat liver beta subunit peptide and its interaction with adenine nucleotides. 290 92

Oligomycin-sensitive particulate ATPase (MB ATPase) from L. donovani promastigotes was solubilized by chloroform treatment. Polyacrylamide gel electrophoresis revealed several protein bands, with the major one possessing ATPase activity. The solubilized enzyme had Mg2+-ATPase and Ca2+-ATPase but no K+-dependent alkaline phosphatase activity. The Mg2+-ATPase activity was stimulated by monovalent cations and was not sensitive to oligomycin. Hence it is referred to as F1 ATPase. It had optimum activity at pH 7.6 and 30 degrees C. The Arrhenius plot for MB ATPase was biphasic with activation energies (Ea) of 16.2 and 3.4 kcal mol-1, while F1 ATPase exhibited a linear plot with Ea = 10.1 kcal mol-1. Lineweaver-Burk plots were biphasic with Km values of 0.17 and 1.25 mM for MB ATPase and 0.18 and 1.33 mM for F1 ATPase. The enzyme could be preserved at -15 degrees C in Tris-SO2-(4)-EDTA-ATP-glycerol (t1/2 = 20 days).
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PMID:Solubilization and kinetic characterization of mitochondrial adenosine triphosphatase from Leishmania donovani promastigotes. 297 May 89

The catalytic properties of the HhaII restriction endonuclease were studied using plasmid pSK11 DNA containing a single 5'-G-A-N-T-C HhaII cleavage site as substrate. Reactions were followed by two methods: 1) gel electrophoretic analysis of nicked circular and linear DNA products, or 2) release of 32P-labeled inorganic phosphate from specifically labeled HhaII sites in a reaction coupled with bacterial alkaline phosphatase. The enzyme is optimally active at 37 degrees C in 10 mM Tris-HCl (pH 9.1) and 4-10 mM MgCl2 without added NaCl. Activity is stabilized by the presence of 2-mercaptoethanol and 0.2% Triton X-100 or 50 microgram/ml bovine serum albumin. At enzyme concentrations below 10 nM and using pSK11 as substrate, initial kinetic rates were dependent on the order of mixing of reactants. A lag of 3-4 min was observed if enzyme or substrate was added last. Preincubation of substrate and enzyme followed by initiation of the reaction with MgCl2 or preincubation of the enzyme with nonspecific DNA followed by initiation with substrate eliminated or reduced the lag, respectively, and speeded up the reactions. Under a wide range of reaction conditions, nicked pSK11 DNA accumulated early, while linear molecules appeared later, suggesting that HhaII cleaves one strand at a time in separate binding events. The apparent Km for covalently closed pSK11 DNA molecules was approximately 17 nM, and the turnover number for the conversion of covalent to nicked sites was 1.1 single strand scissions/min. Pre-steady state kinetic analysis indicated that cleavage of the first phosphodiester bond in a site is first order with a rate constant of about 0.8 min-1, while cleavage of the second phosphodiester bond is first order with a rate constant of about 0.2 min-1.
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PMID:Catalytic properties of the HhaII restriction endonuclease. 299 12

Purified isoenzymes of human alkaline phosphatase from placenta, intestine and liver were investigated as catalysts for phosphotransferase activity, using the phosphoacceptors Tris, 2-amino-2-methyl-1-propanol, 2-amino-2-methyl-1,3-propanediol, diethanolamine, 2-(ethylamino)ethanol, ethanolamine, and N-methyl-D-glucamine. All of the compounds supported phosphotransferase catalysis, conforming to saturation kinetics. There was little difference among the isoenzymes with respect to Km values of the acceptors, but the liver form was the most efficient (highest Vmax/Km) in forming phosphoacceptors; it was also the most efficient (highest Vamax/Ka) when the phosphoacceptors were considered as activators. At Vmax the isoenzymes differed little in their support of phosphotransferase activity relative to phosphohydrolysis, although the intestinal enzyme tended to be the poorest. The two best acceptors were diethanolamine, providing the highest phosphotransferase velocity, and 2-(ethylamino)ethanol, having the lowest Km. The phosphoaceptors that bound Zn2+ tightly did not function well in the phosphotransferase reaction, and vice versa. However, temporal assessment of the phosphohydrolytic and phosphotransferase activities during removal of Zn2+ from the enzyme with 1,10-phenanthroline revealed no evidence of a special role for Zn2+ in the latter activity.
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PMID:Phosphotransferase activity of human alkaline phosphatases and the role of enzyme Zn2+. 303 34

Nourseothricin (streptothricin) causes disturbances (perforations) in the outer membrane of sensitive E. coli strains allowing lysozyme and deoxycholate, but not the periplasmic alkaline phosphatase to penetrate. EDTA slightly increases, but Mg++ ions slightly decrease this effect. The cell walls of three from four nourseothricin-resistant strains do not become permeable under these conditions, but remain sensitive against TRIS/EDTA. Nourseothricin is supposed to pass the outer membrane of sensitive bacteria via some kind of "self-promoting" pathway. This way can (but need not) be blocked in resistant strains.
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PMID:Effect of nourseothricin (streptothricin) on the outer membrane of sensitive and resistant Escherichia coli strains. 304 Sep 61

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.
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PMID:Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase. 307 19

Glial fibrillary acidic protein (GFAP) is soluble in low ionic strength solutions but shows a strong tendency toward assembly with increasing ionic strength as revealed by electron microscopy and turbidity measurements. Increasing K+, Na+, and Li+ concentrations cause an increase followed by a decrease in GFAP turbidity with a maximum at 200 mM, but their effects are much weaker than effects of divalent cations at the same ionic strength. Ca2+, Mg2+, Mn2+, and Ba2+ promote assembly at millimolar concentrations, and 10 microM Cu2+ causes rapid aggregation. The critical concentration for GFAP assembly was 0.08 +/- 0.04 mg/mL in 2 mM Tris-HCl, 60 mM KCl, and 1 mM CaCl2, pH 6.8. The Mr 38,000 rod domain of GFAP obtained by limited chymotryptic digestion is more soluble in 100 mM imidazole hydrochloride buffer, pH 6.8, than the intact molecule, and removal of the end pieces greatly reduces the ability of GFAP to form filaments. BNPS-skatole (2-[(2-nitrophenyl)sulfenyl]-3-methyl-3-bromoindolenine) treatment releases a Mr 30,000 N-terminus and a Mr 20,000 C-terminus. The Mr 30,000 polypeptide shows a higher affinity than the Mr 20,000 fragment for intact GFAP. Arginine and lysine at low concentrations slightly accelerate GFAP assembly, but above 100 mM both amino acids inhibit assembly. ATP, GTP, CTP, and UTP do not show significant effects on GFAP assembly. Dephosphorylation by alkaline phosphatase slightly reduces the assembly ability of GFAP, but phosphatase-treated GFAP still is assembly competent.
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PMID:Factors modulating filament formation by bovine glial fibrillary acidic protein, the intermediate filament component of astroglial cells. 319 99

The mechanism of Tris-BP or Bis-BP (a metabolite of Tris-BP) induced nephrotoxicity was investigated by determining urinary excretion of enzymes and selected metabolites. Rats received single oral doses of 0, 71.7, 143.4 and 286.8 mumol/kg tris (2,3-dibromopropyl) phosphate (Tris-BP) or bis (2,3-dibromopropyl) phosphate (Bis-BP). Urine was collected over a 24 h period and subjected to biochemical examinations. Comparative studies on Tris-BP- and Bis-BP-induced nephrotoxicities were carried out for abnormal patterns of urinary excretion. The urinary excretion of glucose was higher in Bis-BP than Tris-BP at a dose of 143.4 mumol/kg, but this pattern reversed at a dose of 286.8 mumol/kg. Peak lactate excretion occurred later than peak glucose excretion with 143.4 and 286.8 mumol/kg Tris BP and 143.4 mumol/kg Bis-BP. Bis-BP 286.8 mumol/kg caused a transient urinary elevation of lactate on Day 2. Uric acid was excreted at higher levels for Bis-BP than Tris-BP on day 2 of urine collection. Activities of urinary enzymes including alkaline phosphatase, aspartate aminotransferase and gamma-glutamyltransferase, were different on the first day of post-treatment for Tris-BP and Bis-BP. Leucine aminopeptidase and lactate dehydrogenase levels differed on the second day. Activities of the former enzymes on the day 2 urine suggested a transformation of Tris-BP to Bis-BP. Urinary patterns of lactate dehydrogenase isoenzymes (LDH-1-LDH-5) were different between Tris-BP and Bis-BP when rats were treated with the dose of 286.8 mumol/kg: Tris-BP caused a higher excretion of LDH-4 and LDH-5 in urine on day 1 and all five isoenzymes into the day 2 urine. Bis-BP caused slightly higher excretion of LDH-5 and LDH-4 into the day 1 and 3 urine, respectively. Bis-BP but not Tris-BP caused abnormally urinary excretion of sodium ion. Histopathologically, the nephrotoxic effect of Tris-BP appeared one day later and was more obvious than that of Bis-BP in rats after single oral administration.
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PMID:Comparative studies on nephrotoxic effects of tris (2,3-dibromopropyl) phosphate and bis (2,3-dibromopropyl) phosphate on rat urinary metabolites. 335 64


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