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 same isoenzyme of nonspecific alkaline phosphatase (APase), assayed with p-nitrophenylphosphate (p-NPP), was shown be present in different calcifying tissues, bone, calcifying cartilage, odontoblasts and enamel organ. Indications were also found that the enzymatic degradation of inorganic pyrophosphate (PPi) in calcifying tissues is mediated by APase. By using specific APase inhibitors, it was shown that two enzymes capable of degrading ATP exist. These were characterized in dentinogenically active odontoblasts, and it was concluded that one is the classical APase, the other is a Ca2+ and Mg2+ activated ATPase, named Ca2+-ATPase. The two phosphatases were solubilized from odontoblasts and separated. The localization of APase and Ca2+-ATPase in odontoblasts was investigated by subcellular fractionation and EM histochemistry. Routine methods for fixation were found to almost completely inactivate the enzymes. By using a mild fixation technique that preserved 80% of the enzyme activity, the main localization for both APase and Ca2+-ATPase was found to be in the membranes of intercellular vesicles located in the cell body and odontoblasts process. No activity was found in the cell membranes. It is concluded that there are at least two enzymes able to degrade phosphate compounds at alkaline pH in hard tissue forming cells. One is the nonspecific alkaline phosphatase (APase; EC 3. 1. 3. 1), which is active against p-NPP, PPi, glycerophosphates and ATP among other substrates. The other is a more specific Ca2+-ATPase (EC 3. 6. 1. 3). There seems to be an intimate relation between these two enzymes in the tissue. The function of APase in biological calcification is still obscure. In contrast, the finding of an ATP dependent, intravesicularly directed, transmembranous Ca2+-transport in vesicles derived from the microsomal fraction of odontoblasts may explain the role of Ca2+-ATPase.
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PMID:Odontoblast alkaline phosphatases and Ca2+ transport. 15 9

In mineralizing dental tissues the non-specific alkaline phosphatase, using paranitrophenylphosphate (p-NPP) as substrate, is also capable of splitting inorganic pyrophosphate (PPi). In contrast to the p-NPP-ase part of the enzyme, the PPi-ase part requires Zn2+ as a cofactor for its hydrolytic activity. The PPi-ase activity of the enzyme can be inhibited by cadmium ions (Cd2+), perhaps by replacing Zn2+ from the active site of the enzyme molecule. In addition to splitting PPi, the PPi-ase part of the enzyme may also be involved in the phosphorylation process of yet undetermined organic macromolecules. Cd2+ inhibits this phosphorylation process. Inhibition of the PPi-ase activity can also be accomplished by ascorbic acid known for its capacity to complex bivalent cations. Ascorbic acid may accordingly also remove Zn2+ from the active site of the PPi-ase. It is suggested that in developing dental tissues alkaline phosphatase is not only associated with the transport of phosphate ions towards the mineralization front, but is also involved in the phosphorylation of organic macromolecules, a process activated the PPi-ase part of the enzyme.
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PMID:Possible functions of alkaline phosphatase in dental mineralization: cadmium effects. 165 1

p-Nitrophenyl phosphatase (p-NPP-ase) and inorganic pyrophosphatase (PPi-ase) activities originate from the same alkaline phosphatase enzyme. Only the PPi-ase site has zinc (Zn2+) as a cofactor. Cadmium (Cd2+) in concentrations from 10(-5) mol/l upwards inhibited the PPi-ase activity, but did not inhibit the p-NPP-ase activity at all. In mineralizing tooth germs Cd2+ may replace Zn2+, thereby changing the specific stereoconfiguration in the active centre needed for PPi-ase activity, but not that for p-NPP-ase activity.
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PMID:The effects of cadmium on the p-nitrophenyl phosphatase and inorganic pyrophosphatase activities of alkaline phosphatase in developing hamster tooth germs. 255 86

Self recently described a substrate system for alkaline phosphatase (AP)-dependent ELISAs which markedly increased sensitivity, compared to using p-nitrophenyl phosphate. This increase is achieved by having AP, the primary enzyme, produce an activator for a secondary enzyme-substrate system, within which marked amplification occurs. We adapted this technique to study antibodies to casein, bovine serum albumin, ovalbumin, and cardiolipin in the sera of patients with systemic lupus erythematosus (SLE) and normal individuals. The new substrate system yielded titres 30-50-fold higher than those with p-nitrophenyl phosphate (Sigma 104, p-NPP). In addition, when used in a solid-phase C1q binding assay we were able to use a 1 : 100,000 dilution of AP-conjugated anti-human IgG with the amplified substrate, compared to the 1 : 1000 dilution needed with p-NPP. This system is extremely valuable because of its flexibility. It can either be very sparing of limited samples, or if the added sensitivity is not needed, 100-fold less AP conjugate may be used. Thus rare or expensive conjugates can be significantly conserved.
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PMID:A substrate amplification system for enzyme-linked immunoassays. Demonstration of its general applicability to ELISA systems for detecting antibodies and immune complexes. 349 82

Some properties of inorganic pyrophosphatase (PPiase EC 3.6.1.1.) and para-nitrophenylphosphatase (p-NPPase EC 3.1.3.1) in the microsomal fraction of odontoblasts were investigated. The ratio of Mg2+:p-NPP and Mg2+:PPi for optimal enzyme activities was 1:1. A mutual substrate competition for PPiase and p-NPPase was described. In the presence of 0.1 mM EDTA, Mg2+ alone was not able to reactivate p-NPPase or PPiase. Instead, Zn2+ and Co2+ reactivated the PPiase, indicating they might act as cofactors for the enzyme. Mg2+ increased the PPiase activity, probably because Mg PP2-i was the true substrate for the enzyme. The diphosphonates ethane-1-hydroxy 1,1 diphosphonate (EHDP), methane diphosphonate (MDP) and dichloromethane diphosphonate (Cl2MDP) inhibited the PPiase activity.
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PMID:Relationship of inorganic pyrophosphatase and para-nitrophenylphosphatase activities of alkaline phosphatase in the microsomal fraction of isolated odontoblasts. 612 84

We have developed a highly sensitive and quantitative, non-isotopic method of in situ hybridization in which the level of probe binding to intracellular mRNA is determined using an ELISA based detection method. Highly purified cell preparations or cells from a cultured cell line are centrifuged into 96 well microtiter plates. The cells are fixed with formalin and pre-treated with Triton X-100 and Nonidet P40 before photobiotin labeled cDNA probes are applied. The biotin from the hybridization is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and then visualized by the p-NPP (p-nitrophenyl phosphate) conversion method. We have determined a number of the optimal parameters in the procedure including the effects of cell numbers per well, development times and standardization of data using ubiquitous beta-actin mRNA and poly-A+ RNA expression as controls. We have used the technique to study the level of expression of FcgR mRNA in platelets and precursors. We found that platelets and megakaryoblastic cell lines only express mRNA for Fc gamma RII. The presence of the Fc gamma RII molecules was confirmed by complementary studies using immunohistochemistry with specific monoclonal antibodies IV.3 and KB61.
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PMID:Quantitation of Fc gamma RII mRNA in platelets and megakaryoblastic cell lines by a new method of in situ hybridization. 820 59

Nine different isoenzymes and (or) isoforms of alkaline phosphatase (ALP; EC 3.1.3.1) from human tissue were studied with respect to Km and Vmax values for p-nitrophenyl phosphate (p-NPP) in seven different potential phosphoacceptors/buffers. Generally, the phosphoacceptors/buffers with the lowest affinity for p-NPP (highest Km values) gave the highest Vmax values; for the nine enzyme forms in this study, the mean Km and Vmax values were greatest in 2-(ethylamino) (EAE). The two amino-propanol buffers gave the lowest Km and Vmax values. The phosphoacceptors/buffers N-methyl-D-glucamine (MEG), diethanolamine, and Tris had intermediate Km and Vmax values. Hydrophilic liver ALP retained > 90% of its activity after 24 h at 30 degrees C in both 1.0 and 0.3 mol/L Tris and 2-amino-2-methyl-1,3-propanediol and in 0.3 mol/L MEG. This isoenzyme showed greatest inactivation upon prolonged exposure to 1.0 and 0.3 mol/L EAE, the activity at 24 h being approximately 50-66% of that at zero time. p-NPP underwent the greatest spontaneous degradation, approximately 2.5 times that of baseline levels, in 1 mol/L MEG. There was little degradation in all of the buffers tested at 0.3 mol/L or in Tris, EAE, and 2-amino-2-methyl-1-propanol at 1.0 mol/L.
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PMID:Kinetic parameters for the cleaved substrate, and enzyme and substrate stability, vary with the phosphoacceptor in alkaline phosphatase catalysis. 822 23

We have developed an enzyme-linked immunosorbent assay (ELISA) for the sequential analysis of multiple cytokines in limited volumes of biological fluids, including gingival crevicular fluid (GCF) and fibroblast culture supernatants (CS). GCF and CS samples were assayed for multiple cytokines, including IL-1 beta, IL-6, IL-8, GM-CSF and IFN gamma. Immulon 3 microplates were coated with a monoclonal antibody, and a rabbit polyclonal antibody was used to detect the cytokine of interest. Biological samples (200 microL) were added to an anti-IL-1 beta-coated plate and incubated, and 175 microL of each sample were replicate transferred to an anti-IFN gamma-coated plate containing 25 microL/well of diluent. This was repeated in an identical fashion with sequential replicate transfers to an anti-IL-8-coated and finally an anti-IL-6-coated plate. The cytokine standard was a pooled combination of the recombinant human cytokines that were included in the sequence. The plates were developed using an alkaline phosphatase-conjugated goat anti-rabbit IgG and NPP as the substrate. Individual ELISAs ranged in sensitivity from 30 to 2 pg/0.2 mL, with cross-reactivity between these cytokines of < 1%. Additionally, when the same samples were tested in the sequence ELISA vs. the individual ELISA, there was > 85% correlation between the two assays.
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PMID:Sequential ELISA for cytokine levels in limited volumes of biological fluids. 887 92

An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6.5-8.5 were examined using ATP or p-NPP as substrate. At pH 7.4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5-10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.
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PMID:Characterization and cytochemical localization of an ATP diphosphohydrolase from Leishmania amazonensis promastigotes. 1186 92

Kinetic and regulatory properties of the plasma membrane Ca(2+)-ATPase activity from chicken (nucleated) erythrocytes were studied and compared to those from pig (anucleated) erythrocytes. In the absence of known activators: (1) Ca(2+) affinity for the Ca(2+)-ATPase activity from nucleated erythrocytes was 12-fold higher than that from pig erythrocytes, and thus the enzyme is sensitive to physiological Ca(2+) concentrations; (2) the enzyme from chicken erythrocytes showed two apparent Km values for ATP, as compared to one apparent Km value displayed by pig erythrocytes; (3) Ca(2+)-ATPase inserted in chicken erythrocyte membranes showed a low sensitivity to activation by phosphatidylinositol-4-phosphate; (4) when p-NPP was used as substrate, the activity of chicken erythrocytes was high, similar to that attained by pig erythrocytes, but barely sensitive to activation by dimethylsulfoxide and calmodulin. ATP hydrolysis was 10-fold lower than that displayed by pig erythrocytes and the maximal velocity was activated three-fold by calmodulin. The enzyme was insensitive to alkaline phosphatase treatment and showed a single phosphorylation band in electrophoresis, ruling out the possibility of previous modulation by endogenous kinases and/or by partial proteolysis. The differences may be attributed to some endogenous modulator, to distinct isoforms, or to a difference in the E(1)/E(2) states of the enzyme.
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PMID:Regulatory differences between Ca(2+)-ATPase in plasma membranes from chicken (nucleated) and pig (anucleated) erythrocytes. 1197 55


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