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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 inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 381-436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.
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PMID:Kinetics of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide. 881 10

When bovine beta-lactoglobulin (beta-LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhancement of this ligand's fluorescence, was completely recovered. In contrast, the room-temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was approximately 10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (approximately 20 ms). The lability of the freshly refolded protein, as monitored by following the time course of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein. In contrast to the long term postactivation conformational changes detected previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133-1136), we found no changes in either the lability or phosphorescence decays of beta-LG during a period of 24 h. Our results are in agreement with the report by Hattori et al. (1993, J Biol Chem 268:22414-22419), using conformation-specific monoclonal antibodies to recognize native-like structure, that long-term changes occur in the protein conformation, compared with the native structure, on refolding.
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PMID:In vitro renaturation of bovine beta-lactoglobulin A leads to a biologically active but incompletely refolded state. 889 9

The tryptophan residues in green crab (scylla serrata) alkaline phosphatase (EC 3.1.3.1) have been modified by N-bromosuccinimide (NBS). The modification of five tryptophan residues leads to complete loss of enzymatic activity. With the increase of NBS concentration, both the absorption at 278 nm and the fluorescence emission intensity at 335 nm of the modified enzyme decreased markedly indicating the modification of tryptophan residues. Quantitative treatment of the data (Tsou, Sci. Sinica 1962, 11, 1535-1558) shows that among the tryptophan residues modified, one is essential for its catalytic activity. The presence of the substrate markedly protects the modification of tryptophan residues as well as the inactivation, suggesting that the essential tryptophan residue is situated at the active site of this enzyme.
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PMID:An essential tryptophan residue of green crab (syclla serrata) alkaline phosphatase. 913 26

Tissue specimens of squamous cell carcinoma of the larynx from twenty patients were processed for histological and histopathological characterization. A histochemical study of alkaline phosphatase (ALP) was carried out using the simultaneous azo coupling method, and biochemical studies were performed using disodium phenylphosphate as substrate. Full-term, normal placentae were used for comparison. The specific activity of ALP from cancerous laryngeal tissue was 8.9 mKAU/mg protein compared with 154.7 mKAU/mg protein in the placenta. The ALP was localized histochemically in tumor cells (tumor-specific), blood vessels (vascular) and fibrous tissue (interstitial). The tumor-specific phosphatase was sensitive to inhibition by L-phenylalanine, L-leucine and to a lesser degree by L-tryptophan and levamisole. Placental ALP, on the other hand, was completely inhibited by levamisole, more resistant to leucine and more sensitive to phenylalanine and tryptophan. Biochemical estimation of ALP in cancerous laryngeal tissue combined with inhibition studies revealed that the tumor-specific activity of ALP constitutes 15% of the total ALP activity while the major isoenzyme was the vascular ALP, and around one-third of ALP activity was attributed to the interstitial enzyme. The characterization and localization of these isoenzymes are described and compared with that of the placenta. The significance and implications of the above findings are presented.
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PMID:Alkaline phosphatase of cancerous larynx tissue in comparison with the placental enzyme. Biochemical and histochemical studies. 914 Apr 40

The general aromatic amino acid permease, AroP, of Escherichia coli is responsible for the active transport of phenylalanine, tyrosine, and tryptophan. A proposed topological model for the AroP permease, consisting of 12 hydrophobic transmembrane spans connected by hydrophilic loops, is very similar to that of the closely related phenylalanine-specific permease. The validity of this model and its similarity to that of the PheP permease were investigated by studying fusion proteins of AroP permease and alkaline phosphatase. Based on the results obtained from the AroP-alkaline phosphatase sandwich fusions, we have significantly revised the proposed topological model for AroP in two regions. In this modified AroP topological model, the three charged residues E151, E153, and K160 are repositioned within the membrane in span 5. These three residues are conserved in a large family of amino acid transport proteins, and site-directed mutagenesis identifies them as being essential for transport activity. It is postulated that these residues together with E110 in transmembrane span 3 may be involved in a proton relay system.
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PMID:A topological model for the general aromatic amino acid permease, AroP, of Escherichia coli. 915 Feb 30

A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98 degrees C (maximum growth temperature 102 degrees C), but capable of prolonged survival at 105 degrees C. Optimum growth was at pH 7 (range 5-8) and NaCl concentration 2.4% (range 1%-5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn(2+)-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA-DNA hybridization (< 63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp.nov.
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PMID:Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough. 967 87

The major type of acetylcholinesterase in vertebrates (AChET) is characterized by the presence of a short C-terminal domain of 40 residues, the 'tryptophan amphiphilic tetramerization' (WAT) domain. The presence of this domain is not necessary for catalytic activity but is responsible for hydrophobic interactions and for the capacity of AChET subunits to form quaternary associations with anchoring proteins, thereby conditioning their functional localization. In the collagen tail of asymmetric forms, we characterized a small conserved region that is sufficient for binding an AChET tetramer, the proline-rich attachment domain (PRAD). We show that the WAT domain alone is sufficient for association with the PRAD, and that it can attach foreign proteins (alkaline phosphatase, GFP) to a PRAD-containing construct with a glycophosphatidylinositol anchor (GPI), and thus anchor them to the cell surface. Furthermore, we show that isolated WAT domains, or proteins containing a WAT domain, can replace individual AChET subunits in PRAD-linked tetramers. This suggests that the four WAT domains interact with the PRAD in a similar manner. These quaternary interactions can form without intercatenary disulfide bonds. The common catalytic domains of AChE are not necessary for tetrameric assembly, although they may contribute to the stability of the tetramer.
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PMID:A four-to-one association between peptide motifs: four C-terminal domains from cholinesterase assemble with one proline-rich attachment domain (PRAD) in the secretory pathway. 979 27

In order to test the hypothesis (Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, Mellor, Science 281 (1998) 1191-1193) that localized placental tryptophan catabolism prevents immune rejection of the mammalian fetus, the cellular localization and characteristics of human placental indoleamine 2,3-dioxygenase (EC 1.13.11.42) were studied. The localization of indoleamine 2, 3-dioxygenase activity was determined quantitatively using cell fractionation by differential and discontinuous sucrose gradient centrifugation. Enzyme activity was looked for in isolated brush border microvillous plasma membranes of placental syncytiotrophoblast. We found that this membrane preparation (which showed a 32.4-fold purification from the starting homogenate with reference to the activity of a membrane marker enzyme, alkaline phosphatase (EC 3.1.3.1)) was strongly negatively enriched with indoleamine 2,3-dioxygenase (which showed a one twenty-fifth decrease in its specific activity). Placental indoleamine 2, 3-dioxygenase is thus not expressed in the maternal facing brush border membrane of syncytiotrophoblast. 1-Methyl-DL-tryptophan which was used by Munn et al. as a key experimental tool for inhibiting indoleamine 2,3-dioxygenase in the murine model showed a competitive inhibition of human placental indoleamine 2,3-dioxygenase with L-tryptophan. The hypothesis, based on experiments performed in mouse, may therefore be applicable to avoidance of immune rejection of the fetus in human pregnancy.
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PMID:Human placental indoleamine 2,3-dioxygenase: cellular localization and characterization of an enzyme preventing fetal rejection. 1056 24

The room-temperature tryptophan (Trp) phosphorescence lifetime is sensitive to details of the local environment and has been shown to increase significantly in some proteins following H-D exchange. Careful analysis of the phosphorescence lifetime distribution of Trp 109 in Escherichia coli alkaline phosphatase (AP) in solution as a function of time during the H-D exchange shows that this process corresponds to a two-state reaction resulting from the deuteration of a single, specific hydrogen in the core of the protein. The absence of a pH dependence of the exchange rate suggests that the exchange is not an EX2 process, and therefore, a certain degree of unfolding is required for exchange to occur. This discovery opens up the use of phosphorescence-detected hydrogen exchange as a sensitive tool for monitoring the local susceptibility and activation energy for exchange in proteins having a phosphorescent Trp and, for example, for studying the effects of local mutations upon that susceptibility.
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PMID:Hydrogen exchange at the core of Escherichia coli alkaline phosphatase studied by room-temperature tryptophan phosphorescence. 1068 27

Cold-adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three-state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two-state model). The DeltaG for dimer dissociation of cod AP was 8.3 kcal.mol-1, and the monomer stabilization free energy was 2.2 kcal.mol-1, giving a total of 12.7 kcal.mol-1, whereas the total free energy of calf intestinal AP was 17.3 kcal.mol-1. Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate-limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.
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PMID:Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride. 1102 83


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