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)

Human lysosomal arylsulfatase A (ASA) is a prototype member of the sulfatase family. These enzymes require the posttranslational oxidation of the -CH2SH group of a conserved cysteine to an aldehyde, yielding a formylglycine. Without this modification sulfatases are catalytically inactive, as revealed by a lysosomal storage disorder known as multiple sulfatase deficiency. The 2.1 A resolution X-ray crystal structure shows an ASA homooctamer composed of a tetramer of dimers, (alpha 2)4. The alpha/beta fold of the monomer has significant structural analogy to another hydrolytic enzyme, the alkaline phosphatase, and superposition of these two structures shows that the active centers are located in largely identical positions. The functionally essential formylglycine is located in a positively charged pocket and acts as ligand to an octahedrally coordinated metal ion interpreted as Mg2+. The electron density at the formylglycine suggests the presence of a 2-fold disordered aldehyde group with the possible contribution of an aldehyde hydrate, -CH(OH)2, with gem-hydroxyl groups. In the proposed catalytic mechanism, the aldehyde accepts a water molecule to form a hydrate. One of the two hydroxyl groups hydrolyzes the substrate sulfate ester via a transesterification step, resulting in a covalent intermediate. The second hydroxyl serves to eliminate sulfate under inversion of configuration through C-O cleavage and reformation of the aldehyde. This study provides the structural basis for understanding a novel mechanism of ester hydrolysis and explains the functional importance of the unusually modified amino acid.
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PMID:Crystal structure of human arylsulfatase A: the aldehyde function and the metal ion at the active site suggest a novel mechanism for sulfate ester hydrolysis. 952 84

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment. The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile.
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PMID:Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102. 953 86

Cisplatin [cis-dichlorodiammineplatinum (II)] is a widely used chemotherapeutic drug that is toxic to the kidney. Concurrent administration of cysteine together with vitamin E, Crocus sativus and Nigella sativa reduced the toxicity of cisplatin in rats. When administered i.p. for 5 alternate days with 3 mg/kg cisplatin, cysteine (20 mg/kg) together with vitamin E (2 mg/rat) an extract of Crocus sativus stigmas (50 mg/kg) and Nigella sativa seed (50 mg/kg) significantly reduced blood urea nitrogen (BUN) and serum creatinine levels as well as cisplatin-induced serum total lipids increases. In contrast, the protective agents given together with cisplatin led to an even greater decrease in blood glucose than that seen with cisplatin alone. The serum activities of alkaline phosphatase, lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase and alanine aminotransferase of cisplatin-treated rats were significantly decreased, whereas the activities of glutathione reductase and isocitrate dehydrogenase were significantly increased. Addition of cysteine and vitamin E, Crocus sativus and Nigella sativa in combination with cisplatin partially prevented many changes in the activities of serum enzymes. In cisplatin-treated rats, the liver activities of isocitrate dehydrogenase and aspartate aminotransferase were significantly increased, whereas much greater changes were found in the kidneys, with increased activity of glucose-6-phosphate dehydrogenase and decreased activities of alkaline phosphatase, isocitrate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase and gamma-glutamyl transferase, as well as a decreased phosphorylation to oxidation ratio in the mitochondria, indicating reduced adenosine triphosphate production. Also, administration of cysteine and vitamin E, Crocus sativus and Nigella sativa together with cisplatin partially reversed many of the kidney enzymes changes induced by cisplatin. Cysteine together with vitamin E, Crocus sativus and Nigella sativa tended to protect from cisplatin-induced falls in leucocyte counts, haemoglobin levels and mean osmotic fragility of erythrocytes and also prevented the increase in haematocrit. The results of this study indicate a basis for the toxic effects of cisplatin, and suggest a possible way of counteracting the toxicity by introducing protective agents such sulphydryl compounds, other antioxidants and extracts of natural products. It also appears that cells adapt to the effects of cisplatin through the induction of systems that produce NADPH, which in turn compensates the decrease of free sulphydryl groups. We conclude that cysteine and vitamin E, Crocus sativus and Nigella Sativa may be a promising compound for reducing cisplatin-toxic side effects including nephrotoxicity.
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PMID:Protective effect of cysteine and vitamin E, Crocus sativus and Nigella sativa extracts on cisplatin-induced toxicity in rats. 960 69

The membrane topology of the a subunit of the F1F0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin. Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, 176, 196, 238, 241, and 277 (following the COOH-terminal 271 and a hexahistidine tag). None of the single mutations affected the function of the enzyme, as judged by growth on succinate minimal medium. Membrane vesicles with an exposed cytoplasmic surface were prepared using a French pressure cell. Before labeling, the membranes were incubated with or without a highly charged sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. After labeling with the less polar biotin maleimide, the samples were solubilized with octyl glucoside/cholate and the subunit a was purified via the oligohistidine at its COOH terminus using immobilized nickel chromatography. The purified samples were electrophoresed and transferred to nitrocellulose for detection by avidin conjugated to alkaline phosphatase. Results indicated cytoplasmic accessibility for residues 69, 172, 176, and 277 and periplasmic accessibility for residues 8, 24, 27, and 131. On the basis of these and earlier results, a transmembrane topology for the subunit a is proposed.
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PMID:Membrane topology of subunit a of the F1F0 ATP synthase as determined by labeling of unique cysteine residues. 963 82

A close examination of the protein sequence encoded by the Arabidopsis thaliana gene F21M12.26 reveals the gene product to be a phosphomonoesterase, acid optimum (EC 3.1.3.2). A subclass of this broad acid phosphatase is also known as 'histidine acid phosphatase. ' This is the first sequence-based evidence for a 'histidine acid phosphatase' in a dicotyledon. One important member of this class of enzymes is Aspergillus niger (ficuum) phytase, which came into prominence for its commercial application as a feed additive. The putative protein from A. thaliana gene F21M12.26 shares many important features of Aspergillus phytase, namely, size, active-site sequence, catalytic dipeptide and ten cysteine residues located in the key areas of the molecule, but lacks all nine N-glycosylation sites.
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PMID:Identification of a histidine acid phosphatase (phyA)-like gene in Arabidopsis thaliana. 979 Sep 41

A simple separation method enabling the quantification of alkaline phosphatase activity in unfixed, isolated, individual, duodenal epithelial cells has been presented. The activity of intestinal brush border-bound alkaline phosphatase has been demonstrated using naphthol AS-BI phosphate as a substrate and hexazotized New Fuchsin as a simultaneous coupling agent. The amount of final reaction product, as measured cytophotometrically, increases linearly with incubation time (up to 10 min) and with substrate concentration (up to 0.4 mM). Maximum enzyme activity was obtained at pH 8.9. Variation of the substrate concentration revealed the kinetic parameters for naphthol AS-BI phosphate as Km = 0.17 i 0.015 and Vmax = 13.9 +/- 1.38. The specificity of the enzyme reaction was confirmed by the complete inhibition of the enzyme activity in the presence of L-cysteine (10 mm) and 80% inhibition with L-phenylalanine (30 mM). Comparison of alkaline phosphatase activity in 8-microm cryostat sections (beginning at the tip and proceeding to the cryptal part) along the villus axis, with the activity of individual cells obtained by successive separation, revealed similar values of the percentage quotient derived from the entire activities in these two different methods. This suggests that the presented separation procedure gives rise to isolation of the respective cells from the corresponding areas of the villus. Finally, the isolated cells can be used as a valuable tool for the quantitative analysis of alkaline phosphatase activity along the length of the villus.
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PMID:A quantitative histochemical study of alkaline phosphatase activity in isolated rat duodenal epithelial cells. 979 77

Moderate nephrotoxicity was induced in male and female rats exposed to o-xylene for 4 h at atmospheric concentrations of approximately 3000 ppm. The xylene in vivo nephrotoxicity resulted in low enzyme leakage from the kidney into the urine. This low leakage was confirmed in 24-h urine by an increase in gamma-glutamyltranspeptidase (gammaGT), N-acetyl-beta-D-glucosaminidase (NAG) and alkaline phosphatase (ALP) activities. Compared to the control, both the 24-h urine output and the glucose excretion increased in male and female rats. These increases were probably a result of damage to the renal proximal tubules. The role of the metabolic pathway of glutathione in the emergence of the renal damage observed with o-xylene was investigated in rats. Recent studies indicate that the metabolic pathway of glutathione may be a bioactivation pathway, which is responsible for nephrotoxic effects with several drugs or chemicals. The renal toxicity of three synthesized o-xylene thio-conjugates was investigated in several groups of female rats. Administration of S-(o-methylbenzyl)glutathione (i.p., 1 mmol/kg), S-(o-methylbenzyl)cysteine (per os, 1 mmol/kg) or N-acetyl-S-(o-methylbenzyl)cysteine (i.p., 0.75 mmol/kg) to female rats did not induce renal toxicity, as monitored by urinary biochemical parameters (gammaGT, NAG, ALP, glucose). The data obtained suggest that the glutathione pathway would appear to be only detoxication, and probably does not contribute to the renal toxicity of o-xylene in female rats. Thus, either another metabolic pathway or other intermediate metabolites are probably involved in the nephrotoxic action of o-xylene.
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PMID:The role of glutathione and cysteine conjugates in the nephrotoxicity of o-xylene in rats. 980 26

Escherichia coli alkaline phosphatase catalyzes both the nonspecific hydrolysis of phosphomonoesters and a transphosphorylation reaction in which phosphate is transferred to an alcohol via a phosphoseryl intermediate. The rate-determining step for the wild-type enzyme is pH dependent. At alkaline pH, release of the product phosphate from the noncovalent enzyme-phosphate complex determines the reaction rate, whereas at acidic pH hydrolysis of the covalent enzyme-phosphate complex controls the reaction rate. When the lysine at position 328 was substituted with a cysteine (K328C), the rate-determining step at pH 8.0 of the mutant enzyme was altered so that hydrolysis of the covalent intermediate became limiting rather than phosphate release. The transphosphorylation activity of the K328C enzyme was selectively enhanced, while the hydrolysis activity was reduced compared to that of the wild-type enzyme. The ratio of the transphosphorylation to the hydrolysis activities increased 28-fold for the K328C enzyme in comparison with the wild-type enzyme. Several other mutant enzymes for which a positive charge at the active center is removed by site-specific mutagenesis share this characteristic of the K328C enzyme. These results suggest that the positive charge at position 328 is at least partially responsible for maintaining the balance between the hydrolysis and transphosphorylation activities and plays an important role in determining the rate-limiting step of E. coli alkaline phosphatase.
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PMID:Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center. 1005 56

Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximately 9 nM.
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PMID:Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein. 1009 73

The mechanism by which interleukin-1beta (IL-1) inhibits the formation of mineralized tissue nodules by periodontal ligament (PDL) cells in vitro was investigated through the processes of morphological analysis, immunoprecipitation, and Northern blot analysis. PDL cells were obtained from a 2-day-old coagulum in tooth socket and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bone serum (FBS) and antibiotics. Confluent cells were grown for up to 3 weeks in the presence of ascorbic acid (AA), beta-glycerophosphate (GP), and dexamethasone (Dex), or IL-1. PDL cells cultured in the presence of GP and AA did not differentiate, but those treated with Dex, GP, and AA (Dex group) underwent differentiation, showing four stages (confluent, multilayer, nodule, and mineralization) of disparate morphological characteristics. In contrast, the cells treated with IL-1, Dex, GP, and AA (IL-1 group) did form multilayers but failed to form mineralized nodules. Electron microscopy demonstrated that the Dex-induced mineralized nodules contain multilayers of fibroblastic cells, numerous collagen fibrils, and dense globular as well as fused electron dense patches that are associated with numerous apatite crystals. The nodule-like structures in the IL-1 group were also comprised of multilayered fibroblastic cells, but they contained only a small number of collagen fibrils, and no dense globular or fused patches. Von Kossa staining confirmed the presence of numerous mineralized nodules in the Dex group and their scarceness in the IL-1 group. Northern blot analysis of IL-1-treated cells, however, revealed the presence of mRNAs for type I collagen (Col I), secreted protein, acidic and rich in cysteine (SPARC), osteopontin (OPN), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC), whose expression patterns and levels were comparable to those of the Dex group. Immunoprecipitation analysis of OPN and BSP in the cell/matrix layers and the culture media after [35S]-methionine labeling showed their deposition primarily in the mineralized nodules of the Dex group, and their release into the media in the IL-1 group. Immunogold labeling demonstrated the location of OPN and BSP in mineralized nodules of the Dex group, but no significant labeling occurred in the nodule-like structures from the IL-1 group. Interestingly, IL-1 treatment increased the expression of collagenase mRNA by sevenfold, compared with that of the Dex group. These data suggest that the IL-1-induced formation of unmineralized nodules by PDL cells results not so much from the downregulated formation of matrix proteins, which plays a crucial role in the mineralization process, as from their release into the culture media. Finally, collagenase synthesis upregulated by IL-1 may be involved in this process.
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PMID:Interleukin-1beta-induced release of matrix proteins into culture media causes inhibition of mineralization of nodules formed by periodontal ligament cells in vitro. 1020 17


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