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)

Thermal inactivation of alkaline phosphatase of Escherichia coli has been studied at different temperatures (45 to 70 degrees C) and pHs (7.5, 9.0, and 10.0) for the commercial, buffer-dialyzed (pH 9.0) and EDTA-dialyzed (pH 9.0) enzymes. In each case, the inactivation exhibits biphasic kinetics consistent with the rate equation, (formula; see text) where A0 and A are activities at time zero and t, and k1 and k2 are first-order rate constants for the fast and slow phase, respectively. Values of k1 and k2 change independently with temperature, pH, and pretreatment (dialysis) of the enzyme. Time course of inactivation of the enzyme with excess EDTA and effect of Zn2+ ion concentration on the activity of EDTA-dialyzed enzyme have been investigated. The data suggest that the dimeric enzyme protein has two types of catalytic sites which have equal catalytic efficiency (or specific activity) but differ in several other properties. Structural implications of these results have been discussed.
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PMID:Molecular asymmetry in alkaline phosphatase of Escherichia coli. 640 85

Alkaline phosphatase from Escherichia coli contains three metal binding sites (A, B, and C) located at sites forming a triangle with sides of 4, 5, and 7 A (Wyckoff, H.W., Handschumacher, M., Murthy, K., and Sowadski, J.M. (1983) Adv. Enzymol. 55, 453). When all three sites are occupied by Cd(II) the enzyme has a very low turnover; at least 10(3) slower than the native Zn(II) enzyme. The slow turnover number has made the Cd(II) enzyme useful in NMR studies of the mechanism of alkaline phosphatase. The binding of arsenate to two forms of Cd(II) alkaline phosphatase (Cd(II)2alkaline phosphatase and Cd(II)6alkaline phosphatase) has been studied by 113Cd NMR. Cd(II)2alkaline phosphatase, pH 6.3, binds arsenate at only one monomer of the dimeric enzyme and causes migration of Cd(II) from the A site of one monomer to the B site of the arsenylated monomer. This same migration has previously been observed to accompany metal ion-dependent phosphate binding, but is much more rapid in the case of arsenate. The acceleration of migration induced by arsenate supports the conclusion based on the phosphate data that the substrate anion binds to the A site metal ion of one monomer prior to migration and that only the metal ion at A site is required for phosphorylation (arsenylation) of serine 102. The 113Cd chemical shifts of A and B site metal ions are very sensitive to the form of the bound arsenate, i.e. covalent (E-As) or noncovalent (E X As) complex. Like the analogous phosphate derivatives, the change of chemical shift of A site (to which phosphate is coordinated in the E X P complex) is much greater than that of the B site metal ion, when the arsenate shifts between the two intermediates, suggesting that arsenate is also coordinated to A site in the E X As intermediate. The chemical shifts of A and B site 113Cd(II) ions are considerably different in the arsenate and phosphate derivatives, while the C site 113Cd(II) ions have nearly identical chemical shifts. Thus the substrate appears to interact closely with both A and B sites, while C site appears relatively unimportant in phosphomonoester hydrolysis. The analogous behavior of arsenate and phosphate at the active center as evaluated by 113Cd NMR supports the validity of using the heavier arsenate derivative in x-ray diffraction studies.
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PMID:113Cd NMR. Arsenate binding to Cd(II) alkaline phosphatase. 642 81

1,25-Dihydroxycholecalciferol increases the incorporation of leucine into a protein component (Mr = 84000) of the chick intestinal brush-border membrane. Evidence has now been obtained showing this protein to be sub-unit of alkaline phosphatase. Pure chick intestinal alkaline phosphatase was prepared and antibodies raised against it in rabbits. This enzyme and the brush-border protein behaved identically on gel electrophoresis, and in terms of their phosphorylating activity and enzyme activity whether as the enzymically active dimeric form or when reduced to the sub-unit form. The anti-(alkaline phosphatase) reacted with the protein into which the incorporation of leucine was stimulated by 1,25-dihydroxycholecalciferol.
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PMID:Incorporation of labelled leucine into alkaline phosphatase in response to 1,25-dihydroxycholecalciferol in chick intestinal brush borders. 689 55

Carbon-13 nuclear magnetic resonance (13C NMR) of Escherichia coli alkaline phosphatase labeled biosynthetically with beta,beta-[gamma-13C]dideuteriohistidine has been used to determine the number and identity of the histidine residues that participate in metal ion coordination at the three classes of binding sites in this dimeric Zn2+ metalloenzyme. Detailed 13C NMR titrations of the apoenzyme with 113Cd2+ and Mg2+, in conjunction with parallel 13 Cd NMR measurements [Otvos, J.D., & Armitage, I.M. (1980) Biochemistry (third of three papers in this issue)], permitted the assignment of four histidine residues as ligands to the "catalytic", or A site, metal ions, two coordinated via their N pi imidazole nitrogens and two via N pi. In addition, a fifth histidyl ligand, coordinated through N pi, was shown to be located at the "structural", or B, sites on the dimer. The "regulatory", or C, sites do not contain histidyl metal ligands. Unambiguous identification of the three histidines coordinated to metal ion via N pi was provided by the observation of resolved 113Cd-13C spin-spin coupling (3J = 12-19 Hz) in their gamma-carbon resonances. Once assigned, the 13C resonances of the five histidyl metal ligands were used to monitor the relative affinities of the A, B, and C sites for Cd2+ and Zn2+. At pH 6.3, Cd2+ was found to bind to the A sites at least 10 times tighter than to the B or C sites, which have roughly equal affinities. In marked contrast, Zn2+ was found to have similar affinities for the A and B sites at both pH 6.3 and 8.0. The affinity of the C sites for Zn2+ and Mg2+ was shown to be at least an order of magnitude lower. The binding constants of all three sites for Cd2+ and Zn2+ are greater than 10(5) M-1. Evidence is also presented that suggests that the A, B, and C sites may be located in close proximity to one another in the monomers.
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PMID:Characterization of the properties of the multiple metal binding sites in alkaline phosphatase by carbon-13 nuclear magnetic resonance. 699 14

Cadmium-113 nuclear magnetic resonance (113Cd NMR) has been used to probe the binding characteristics of 113Cd2+ to the three classes of metal binding sites in Escherichia coli alkaline phosphatase to help elucidate the molecular origin of the metal ion dependent "half-sites" reactivity exhibited by this dimeric Zn2+ metalloenzyme [Otvos, J.D., Armitage, I.M., Chlebowski, J.F., & Coleman, J.E. (1979) J. Biol. Chem. 254, 4707-4713]. In the absence of phosphate, the first two 113Cd2+ ions added to the apodimer give rise to a single 113Cd resonance (169 ppm), indicating selective binding to the pair of symmetrically disposed A sites. Resonances arising from additional 113Cd2+ bound to the B and C sites cannot be observed; B- and/or C-site occupation also renders the A-site 113Cd resonance undetectable. Both these observations have been attributed to severe chemical exchange broadening in the A-, B-, and C-site 113Cd signals induced by an unknown modulation process(es). Interestingly, covalent phosphorylation of the active-site serine residues abolishes this exchange modulation, allowing three separate resonances to be detected and assigned to 113Cd2+ located at each of the three classes of metal binding sites in the enzyme. By varying the metal composition of the phosphorylated enzyme, we have characterized the correlations that exist between the chemical shifts ana intensities of these 113Cd resonances and the metal occupancies of the A, B, and C sites in the individual subunits. This information has allowed us to conclude that the half-sites phosphorylation of the Cd2 2+ enzyme is accompanied by a slow migration of half the Cd2+ originally located at the A sites to the B sites on the phosphorylated subunits. The driving force for this metal redistribution, which at equilibrium leaves half the subnits devoid of metal ion and thereby incapable of binding phosphate, is apparently the dramatic stabilization of the complex of Cd2+ with the B sites, which was demonstrated to occur in those subunits that become phosphorylated. From the kinetics of both phosphorylation and metal redistribution in Cd2 2+ enzyme, we suggest that population of the A and B sites in a subunit, rather than the A site alone, constitutes the minimum requirement for induction of catalytic function in alkaline phosphatase. The spin relaxation properties of the enzyme-bound 113Cd2+ ions are also briefly discussed.
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PMID:Determination by cadmium-113 nuclear magnetic resonance of the structural basis for metal ion dependent anticooperativity in alkaline phosphatase. 699 15

Chemical modification of Escherichia coli alkaline phosphatase using the group-specific reagent, ethoxyformic anhydride, has demonstrated that 3 histidyl residues/subunit are modified with a concomitant loss of enzyme activity. Reaction with [14C]ethoxyformic anhydride indicates that only three ethoxyformyl groups are incorporated per subunit, confirming that no other amino acid residues are modified under these conditions. Zinc ions protect alkaline phosphatase from inactivation as well as from histidine modification, thus implicating all 3 histidyl residues in Zn2+ binding. The ethoxyformylation reaction was also used to characterize Zn2+ binding sites in immobilized dimeric and monomeric alkaline phosphatase derivatives. The immobilized dimeric alkaline phosphatase was inactivated with ethoxyformic anhydride at a rate similar to that of the soluble enzyme, demonstrating that immobilization did not significantly alter the chemical environment of the Zn2+ binding site. The catalytically inactive, immobilized monomer of alkaline phosphatase was modified more rapidly with ethoxyformic anhydride, demonstrated by the loss of its ability to form functionally active enzyme upon titration with nascent soluble subunits. Moreover, Zn2+ protects the immobilized subunit alkaline phosphatase against this modification, indicating that the isolated subunits of alkaline phosphatase bind Zn2+. These results are consistent with a model for renaturation of the dimeric enzyme in which individual subunits refold and bind Zn2+ before which individual subunits refold and bind Zn2+ before establishing subunit interactions to regain catalytic activity.
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PMID:Evidence for histidyl residues at the Zn2+ binding sites of monomeric and dimeric forms of alkaline phosphatase. 701 46

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

We report the production in Escherichia coli of a murine antibody IgG2b, a murine::human chimeric antibody IgG3 and the corresponding F(ab')2 fragments, all directed against human placental alkaline phosphatase, a tumor marker. The cDNA of the heavy chain of the mature antibodies and their fragments were linked up to the bacterial alkaline phosphatase signal sequence and were placed under control of the inducible tac promoter. Coexpression with the murine kappa light chain resulted in production of functional dimeric, trimeric and tetrameric, mature antibodies and F(ab')2 fragments in the periplasm of E. coli in a yield of 200-300 micrograms l-1. High amounts of light and heavy chains were present also in the insoluble fraction.
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PMID:Production in Escherichia coli of a functional murine and murine::human chimeric F(ab')2 fragment and mature antibody directed against human placental alkaline phosphatase. 757 33

We describe the preparation of a new rat monoclonal antibody (CRC1) to the N-terminal sequence of the 43 kDa subunit of human ovarian inhibin, and its use together with other anti-peptide monoclonal antibodies, in two-site immunoassays for the detection of inhibin-related material in biological fluids. The Fab fraction of a mouse monoclonal antibody (R1) to the N-terminal portion of the 20 kDa alpha subunit, coupled to alkaline phosphatase, was used for detection, and either CRC1 or a monoclonal antibody (E4) to the beta-A subunit were used as capture antibodies. The E4/R1 combination, expected to measure dimeric bioactive inhibin, could detect less than 2 pg/ml of recombinant inhibin in diluent, gave good recovery of activity spiked into human blood, and could measure significant levels of immunoreactivity in sera from women undergoing ovulation induction, and in some normal women. Sera from post-menopausal women contained undetectable levels. Apparent inhibin levels in human follicular fluid were increased six-fold by pretreatment with 8 M urea, suggesting masking of epitopes in this fluid. Activin cross-reactivity in the assay was 0.05%. The R1/CRC1 assay, expected to measure only large molecular weight forms of inhibin or its alpha subunit, could detect immunoreactivity in human FF diluted 50,000-fold, and in all sera tested, although the levels in the hyperovulated women were higher. By contrast to the E4/R1 assay much of the immunoreactivity was labile during the clotting process, or subsequent assay, and reliable measurements on blood with this assay will require special sample collection procedures. These results demonstrate the value of anti-peptide monoclonal antibodies in the study of inhibin, and the results obtained with CRC1 show that antibodies useful for immunoassays can sometimes be obtained without the purified target molecule being available for immunization or screening.
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PMID:Immunoassays for inhibin and its subunits. Further applications of the synthetic peptide approach. 769 20

Alkaline phosphatase (ALP, EC 3.1.3.1) is a membrane-bound metalloenzyme that consists of a group of true isoenzymes, all glycoproteins, encoded for by at least four different gene loci: tissue-nonspecific, intestinal, placental, and germ-cell ALP. Through posttranslational modifications of the tissue-nonspecific gene, for example, through differences in carbohydrate composition, bone and liver ALP are formed. Nowadays, most commercially available methods for separating or measuring ALP isoenzymes are easy to perform and sensitive and allow for reproducible and quantitative results. As more isoenzymes and isoforms have been characterized, confusion has arisen due to the many different names they were given. For the sake of simplicity and because of structural analogies, we propose an alternative nomenclature for the ALP isoenzymes and isoforms based on their structural characteristics: soluble, dimeric (Sol), anchor-bearing (Anch), and membrane-bound (Mem) liver, bone, intestinal, and placental ALP. Together with lipoprotein-bound liver ALP and immunoglobulin-bound ALP, these names largely fit the many forms of ALP one can encounter in human serum and tissues. The clinically relevant isoenzymes are sol-liver, Mem-liver, lipoprotein-bound liver, and Sol-intestinal ALP in liver diseases, and Sol-bone and Anch-bone ALP in bone diseases. Many different isoenzyme patterns can be found in malignancies and renal diseases. This test provides the clinician with valuable information for diagnostic purposes as well as for follow-up of patients and monitoring of treatment. However, ALP isoenzyme determination will only provide clinically useful information if the patterns are correctly interpreted. In this respect, care should be taken to use the proper reference ranges, taking into account the age and sex of the patient. A normal total ALP activity does not rule out the presence of an abnormal isoenzyme pattern, particularly in children. Separating ALP into its isoenzymes adds considerable value to the mere assay of total ALP activity.
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PMID:Interpretation and clinical significance of alkaline phosphatase isoenzyme patterns. 781 74


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