Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

19F nuclear magnetic resonance (NMR) spectroscopy has been used to study a fully active E. coli fluorotyrosine alkaline phosphatase. The fluorotyrosine resonances provide sensitive probes of the conformational states of the protein. They were used to follow the addition of zinc or cobalt to the apoprotein, and the titration of the protein with inorganic phosphate or the inhibitor 2-hydroxy-5-nitrobenzylphosphonate. The results indicate that 2 molecules of inorganic phosphate per dimer of alkaline phosphatase are required to complete a general conformational change in the protein involving perturbations to the environment of several tyrosines. Spectra of the cobalt enzyme indicate that on specific tyrosine per subunit may be near the metal site. The 19F NMR results, combined with the 31P NMR results in the accompanying paper, lead directly to the conclusion that dissociation of noncovalently bound inorganic phosphate from the enzyme is the rate-limiting process in enzyme catalysis at high pH. The local environment of the individual fluorotyrosines is also discussed.
 ...
PMID:Fluorine-19 nuclear magnetic resonance study of fluorotyrosine alkaline phosphatase: the influence of zinc on protein structure and a conformational change induced by phosphate binding. 0 91

Ultraviolet difference spectra are produced by the binding of divalent metal ions to metal-free alkaline phosphatase (EC 3.1.3.1). The interaction of the apoprotein with Zn2+, Mn2+, Co2+ and Cd2+, which induce the tight binding of one phosphate ion per dimer, give distinctly different ultraviolet spectra changes from Ni2+ and Hg2+ which do not induce phosphate binding. Spectrophotometric titrations at alkaline pH of various metallo-enzymes reveal a smaller number of ionizable tyrosines and a greater stability towards alkaline denaturation in the Zn2+- and Mn2+-enzymes than in the Ni2+-, Hg2+- and apoenzymes. The Zn2+- and Mn2+-enzymes have CD spectra in the region of the aromatic transitions that are different from the CD spectra of the Ni2+-, Hg2+- and apoenzymes. Modifications of arginines with 2,3-butanedione show that a smaller number of arginine residues are modified in the Zn2+-enzyme than in the Hg2+-enzyme. The presented data indicate that alkaline phosphatase from Escherichia coli must have a well-defined conformation in order to bind phosphate. Some metal ions (i.e. Zn2+, Co2+, Mn2+ and Cd2+), when interacting with the apoenzyme, alter the conformation of the protein molecule in such a way that it is able to interact with substrate molecules, while other metal ions (i.e. Ni2+ and Hg2+) are incapable of inducing the appropriate conformational change of the apoenzyme. These findings suggest an important structural function of the first two tightly bound metal ions in enzyme.
 ...
PMID:Metal ion-induced conformational changes in Escherichia coli alkaline phosphatase. 1 23

5'-Nucleotidase, assayed as 5'-AMPase, has been extensively characterized and established as a stable, quantitative plasma membrane marker in HeLa S3 cells. The membrane 5'-AMPase has a Km of 7.0 microM. Relative affinities of the other 5'-mononucleotides for the enzyme are 5'-GMP > 5'-TMP > 5'-UMP > 5'-CMP. There are activity optima at pH7 and 10; the latter is Mg(2+)-dependent. The membrane preparations have a small amount of acid phosphatase activity that is distinct from 5'-AMPase activity but no alkaline phosphatase. AOPCP, ADP, and ATP are strongly inhibitory. Mg2+, Ca2+, or Co2+ additions do not affect the pH 7.0 activity; Mn2+ activates slightly, whereas Zn2+, Cu2+, and Ni2+ are inhibitory. EDTA slowly inactivates, but removal of the EDTA without the addition of divalent cations restores activity. The inactivation is also substantially reversed by Co2+ or Mn2+, but reactivability by divalent cations decreases with time in EDTA. ConA strongly inhibits, and alpha-methyl-D-mannoside or glucose (the latter much less efficiently) relieves the inhibition, indicating that the 5'-AMPase is a glycoprotein. Histidine is also inhibitory. Ouabain, phloretin, cytochalasin B, cysteine, phenyl-alanine, MalNEt, and IAA are without effect. 5'-AMPase activity codistributes with pulse-bound [3H]ouabain when either of two cell fractionation procedures are used. The 5'-AMPase activity per cell is constant at different cell densities in exponentially growing cells, and activity per unit cell volume remains constant throughout the cell cycle. These properties, together with its absence in other organelles, its stability to storage, its insensitivity to certain experimental manipulations, and its general insensitivity to inhibitors of specific transport systems, make 5'-AMPase a useful quantitative marker in studies on the regulation of HeLa membrane transport systems. Key Words: HeLa, 5'-nucleotidase, plasma membrane marker, non-specific phosphatases, divalent ions, ConA, AOPCP, cell cycle, mitochondria, transport inhibitors.
 ...
PMID:Characterization of HeLa 5'-nucleotidase: a stable plasma membrane marker. 4 80

Nonspecific alkaline phosphatase activity was identified in human normal and diseased breasts with the use of the calcium-cobalt, the lead-nitrate, and the azo-dye methods. The results varied not only with the staining method, but also with the functional status of the breast structures. In normal, dysplastic, and fibroadenomatous tissues there was a strong parallelism between myoepithelial and capillary enzyme activities. The calcium-cobalt method was the only technique which allowed staining of carcinoma cells; cancer stromal enzyme activity was evidenced only with the use of the same method. Our findings suggest that nonspecific alkaline phosphatase activity probably reflects a functional status of the labelled structures; the enzyme activity of myoepithelial cells is variable and not really specific.
 ...
PMID:Nonspecific alkaline phosphatase activity in normal and diseased human breast. 11 21

The conditions necessary for the secretion of phospholipase C (phosphatidylcholine cholinephosphohydrolase) by Pseudomonas aeruginosa were studied. Enzyme secretion by washed cell suspensions required a carbon source and ammonium, potassium, and calcium ions. The calcium requirement could be substituted by magnesium and strontium but not by copper, manganese, cobalt, or zinc. During growth in liquid medium, cells secreted phospholipase C during late logarithmic and early stationary phases. Secretion was repressed by the addition of inorganic phosphate but not by organic phosphates, glucose, or sodium succinate. Studies with tetracycline indicated that de novo protein synthesis was necessary for the secretion of phospholipase C and that the exoenzyme was not released from a preformed periplasmic pool. Similarly, extraction of actively secreting cells with 0.2 M MgCl2 at pH 8.4 solubilized large quantities of the periplasmic enzyme alkaline phosphatase but insignificant amounts of phospholipase C. Bacteria continued to secrete enzyme for nearly 45 min after the addition of inorganic phosphate or rifampin.
 ...
PMID:Secretion of phospholipase C by Pseudomonas aeruginosa. 11 87

The binding of metal to alkaline phosphatase from Escherichia coli and the binding of water and orthophosphate to the Me-2+-enzyme binary complex have been examined by water proton relaxation rate (PRR) measurements. Titration of the three paramagnetic metals, Mn2+, Cu2+, and Co2+, into apoalkaline phosphatase and the titrations of apoenzyme into metal have been carried out. Analysis of the spin-lattice relaxation rates for these titrations and of Scatchard binding curves derived from these results, as well as EPR data, show four tight manganese sites, between two and three tight copper sites, or four cobalt sites per enzyme dimer of molecular weight 80,000. The multiple sites for each metal are indistinguishable by these magnetic resonance techniques. Both the spin-lattice- and spin-spin-relaxation rates exhibit a negative temperature coefficient, showing that these processes are not exchange-limited. From a frequency dependence study of T-1 and from the T-1:T-2 ratio measured at 220 MHz, correlation times from the water-enzyme complexes have been estimated. For H20-Mn-2+-alkaline phosphatase, gamma c equals 1.55 times 10-9 s; for H20-Cu-2+ -alkaline phosphatase, gamma c equals 1.82 times 10-s; and for the cobalt complex, gamma c equals 1.0 times 10-12 s at 4 degrees. Assuming 1 water molecule bound per metal site, these correlation times correspond to the following water-metal distances: gamma (A) is 4.0 A for Mn-2+-H20, 3.4 A for Cu-2+-H20, and 2.8 A for Co-2+-H20. Thus, water is shown to bind directly to the metal atoms of alkaline phosphatase. The correlation between the length of the water-metal bond and the relative activity of the various metalloenzymes support the importance of this binding in the monophosphoesterase reaction catalyzed by alkaline phosphatase. Addition of excess orthophosphate to any of the water-metalloenzyme complexes does not displace an exchangeable water molecule from the metal site. The Mn-PO-4 distance which we have reported earlier (Zukin, R.S., Hollis, D.P., and Gray, G.A. (1973) Biochem. Biophys. Res. Commun. 53, 238) to be 7.3 A is consistent with this finding and suggests a model in which Pi binds to Mn-2+-alkaline phosphatase through a water bridge.
 ...
PMID:Role of metal ions in Escherichia coli alkaline phosphatase. A study of the metal-water interaction by nuclear relaxation rate measurements on water protons. 16 41

To facilitate the study of individual metal binding sites of polymeric metalloproteins, conversion of exchange-labile Co(II) in E. coli alkaline phosphatase (EC 3.1.3.1) to exchange-inert Co(III) was examined. Oxidation of Co(II) alkaline phosphatase with hydrogen peroxide results in a single absorption maximum at 530 nm and loss both of the characteristic electron paramagnetic signal and of enzymatic activity. Zinc neither reactivates this enzyme nor displaces the oxidized cobalt atoms. Metal and amino-acid analyses demonstrate that oxidation alters neither cobalt binding nor amino-acid composition of the enzyme. Al data are consistent with the conclusion that hydrogen peroxide oxidizes Co(II) in alkaline phosphatase to Co(III). Polymeric metalloenzymes can contain different categories of metal atoms serving in catalysis, structure stabilization, and/or control and exerting their effects independently or interdependently. The in situ conversion of exchange-labile Co(II) to exchange-stable (Co(III) offers a method to selectively and differentially "freeze" cobalt atoms at their respective binding sites. The accompanying spectral changes and concomitant retardation in ligand exchange reactions may be used to differentiate between specific metal binding sites that serve different roles in polymeric metalloenzymes.
 ...
PMID:Cobalt(III), a probe of metal binding sites of Escherichia coli alkaline phosphatase. 16 26

A metal-ion-independent, nonspecific phosphoprotein phosphatase (Mr = 35000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p-nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ greater than Mn2+ greater than Co2+) and a sulfhydryl compound for activity, has been found to co-purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal-ion-dependent form which, subsequently, can be reactivated by Co2+ greater than Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented.
 ...
PMID:Purification and properties of a phosphorylase (phosphoprotein) phosphatase associated with an alkaline phosphatase of Mr 35000 from bovine adrenal cortex. 23 Sep 63

The magnetic circular dichroic (MCD) spectra of cobalt(II) sugstituted metalloenzymes have been studied and compared to a series of four-, five-, and six-coordinate cobalt(II) model complexes previously examined (T. A. Kaden et al. (1974), Inorg. Chem. 13, 2582). The MCD spectra of cobalt substituted carboxypeptidase A, procarboxypeptidase ta, and thermolysin are consistent with earlier deductions of tetrahedral coordination from absorption spectra and also with X-ray structure analysis. Inhibitors fail to alter their MCD spectra significantly. The MCD spectra of cobalt alkaline phosphatase and carbonic anhydrase are more complex and their pH dependence and alteration by inhibitors are discussed in terms of known cobalt(II) models.
 ...
PMID:Magnetic circular dichroic spectra of cobalt(II) substituted metalloenzymes. 23 52

Extracts of adult Paramphistomum explanatum have been shown to contain high concentration of acid phosphomonoesterase with maximum activity at pH 4.5. The enzyme has been characterized by an exhibition of an unexpected increase in the inhibitory action of a mercury at 1 mM concentration by EDTA. With a lower concentration of mercury (0.1 mM and below) EDTA gave partial protection against inhibition. Different concentrations of magnesium and cobalt activated the enzyme while fluoride, copper, arsenate, tartrate and p-mercuribenzoate brought about inhibition. EDTA, glycine, glutathione and sodium azide had no effect. There was an indication of the presence of alkaline phosphomonoesterase at pH 10.0. The Km for p-nitrophenyl phosphate hydrolysis was 0.45 mM at pH 4.5.
 ...
PMID:Phosphatase systems in Paramphistomum explanatum Fischoeder, 1901. 23 47


1 2 3 4 5 6 7 8 9 10 Next >>