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)

Kinetic evidence for the role of divalent metal ions in the phosphotransferase activity of polidocanol-solubilized alkaline phosphatase from osseous plate is reported. Ethylenediamine tetreacetate, 1,10-phenanthrolin, and Chelex-100 were used to prepare metal-depleted alkaline phosphatase. Except for Chelex-100, either irreversible inactivation of the enzyme or incomplete removal of metal ions occurred. After Chelex-100 treatment, full hydrolase activity of alkaline phosphatase was recovered upon addition of metal ions. On the other hand, only 20% of transferase activity was restored with 0.1 microM ZnCl2, in the presence of 1.0 M diethanolamine as phosphate acceptor. In the presence of 0.1 mM MgCl2, the recovery of transferase activity increased to 63%. Independently of the phosphate acceptor used, the transferase activity of the metal-depleted alkaline phosphatase was fully restored by 8 microM ZnCl2 plus 5 mM MgCl2. In the presence of diethanolamine as phosphate acceptor, manganese, cobalt, and calcium ions did not stimulate the transferase activity. However, manganese and cobalt-enzyme catalyzed the transfer of phosphate to glycerol and glucose.
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PMID:Dependence of divalent metal ions on phosphotransferase activity of osseous plate alkaline phosphatase. 917

Particulate wear debris can induce the release of bone-resorbing cytokines from cultured macrophages and fibroblasts in vitro, and these mediators are believed to be the cause of the periprosthetic bone resorption which leads to aseptic loosening in vivo. Much less is known about the effects of particulate debris on the growth and metabolism of osteoblastic cells. We exposed two human osteoblast-like cell lines (SaOS-2 and MG-63) to particulate cobalt, chromium and cobalt-chromium alloy at concentrations of 0, 0.01, 0.1 and 1.0 mg/ml. Cobalt was toxic to both cell lines and inhibited the production of type-I collagen, osteocalcin and alkaline phosphatase. Chromium and cobalt-chromium were well tolerated by both cell lines, producing no cytotoxicity and no inhibition of type-I collagen synthesis. At the highest concentration tested (1.0 mg/ml), however, chromium inhibited alkaline phosphatase activity, and both chromium and cobalt-chromium alloy inhibited osteocalcin expression. Our results clearly show that particulate metal debris can modulate the growth and metabolism of osteoblastic cells in vitro. Reduced osteoblastic activity at the bone-implant interface may be an important mechanism by which particulate wear debris influences the pathogenesis of aseptic loosening in vivo.
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PMID:The effects of particulate cobalt, chromium and cobalt-chromium alloy on human osteoblast-like cells in vitro. 976 14

Retention of iron, copper and cobalt in the guinea pig organism as affected by imuran increases while that of zinc sharply decreases. These bioelements are considerably redistributed in the blood, skin, liver, muscles, bones, spleen; activity of metalloenzymes conjugated with them (lactate dehydrogenase, carboanhydrase, alkaline phosphatase, ceruloplasmin) in the blood changes.
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PMID:[Effect of imuran on the activity of metalloenzymes and parameters of metal metabolism in the body]. 929 98

A hyperthermophilic alkaline phosphatase was purified from Thermotoga neapolitana by heat treatment at 100 degrees C in the presence of Co2+ followed by ion-exchange and affinity chromatographies. The enzyme was purified 2,880-fold with 44% yield. The purified enzyme showed a single protein band of M(r) 45,000 on SDS-PAGE and an apparent molecular weight of 87,000 estimated by gel filtration chromatography. This suggested a homogenous dimer structure. The optimal pH and temperature for enzyme activity were 9.9 and 85 degrees C, respectively. Under optimal conditions, T. neapolitana alkaline phosphatase displayed 30% higher activity than calf intestine alkaline phosphatase did with p-nitrophenyl-phosphate as substrate. The hyperthermostable enzyme had a half-life of 238 min at 90 degrees C and K(m) and Vmax values of 183 microM and 1,352 U mg-1, respectively. Co2+ enhanced the enzyme activity, thermostability, and ligand affinity during column chromatography. The alkaline phosphatase was twice as active with Co2+ than with either Zn2+ or Mn2+ as the metal cofactor.
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PMID:Purification and characterization of alkaline phosphatase from Thermotoga neapolitana. 932 73

Rat osseous plate alkaline phosphatase is a metalloenzyme with two binding sites for Zn2+ (sites I and III) and one for Mg2+ (site II). This enzyme is stimulated synergistically by Zn2+ and Mg2+ (Ciancaglini et al., 1992) and also by Mn2+ (Leone et al., 1995) and Co2+ (Ciancaglini et al., 1995). This study was aimed to investigate the modulation of enzyme activity by Ca2+. In the absence of Zn2+ and Mg2+, Ca2+ had no effects on the activity of Chelex-treated, Polidocanol-solubilized enzyme. However, in the presence of 10 microM MgCl2, increasing concentration of Ca2+ were inhibitory, suggesting the displacement of Mg2+ from the magnesium-reconstituted enzyme. For calcium-reconstituted enzyme, Zn2+ concentrations up to 0.1 microM were stimulatory, increasing specific activity from 130 U/mg to about 240 U/mg with a K0.5 = 8.5 nM. Above 0.1 microM Zn2+ exerted a strong inhibitory effect and concentrations of Ca2+ up to 1 mM were not enough to counteract this inhibition, indicating that Ca2+ was easily displaced by Zn2+. At fixed concentrations of Ca2+, increasing concentrations of Mg2+ increased the enzyme specific activity from 472 U/mg to about 547 U/mg, but K0.5 values were significantly affected (from 4.4 microM to 38.0 microM). The synergistic effects observed for the activity of Ca2+ plus magnesium-reconstituted enzyme, suggested that these two ions bind to the different sites. A model to explain the effect of Ca2+ on the activity of the enzyme is presented.
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PMID:Effect of calcium ions on rat osseous plate alkaline phosphatase activity. 933 71

The membrane-bound CzcCBA protein complex mediates heavy metal resistance in Alcaligenes eutrophus by an active cation efflux mechanism driven by cation-proton antiport. The CzcA protein alone is able to mediate weak resistance to zinc and cobalt and is thus the central antiporter subunit. The two histidine-rich motifs in the CzcB subunit are not essential for zinc resistance; however, deletion of both motifs led to a small but significant loss of resistance to this cation. Translation of the czcC gene encoding the third subunit of the CzcCBA complex starts earlier than predicted, and CzcC is probably a periplasmic protein, as judged by the appearance of two bands after expression of czcC in Escherichia coli under control of the phage T7 promoter. Fusions of CzcC and CzcB with alkaline phosphatase and beta-galactosidase are in agreement with a periplasmic location of most parts of both proteins. Both CzcC and CzcB are bound to a membrane, probably the outer membrane, by themselves and do not need either CzcA or each other as an anchoring protein. Based on these data, a new working model for the function of the Czc system is discussed.
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PMID:New functions for the three subunits of the CzcCBA cation-proton antiporter. 937 29

Microsomal fractions from pig and calf brain catalyze the enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate (Dol-P) (Sumbilla, C. A., and Waechter, C. J. (1985) Methods Enzymol. 111, 471-482). The Dol-P phosphatase (EC 3.1.3.51) has been solubilized by extracting pig brain microsomes with the nonionic detergent Nonidet P-40 and purified approximately 1,107-fold by a combination of anion exchange chromatography, polyethylene glycol fractionation, dye-ligand chromatography, and wheat germ agglutinin affinity chromatography. Treatment of the enzyme with neuraminidase prevented binding to wheat germ agglutinin-Sepharose, indicating the presence of one or more N-acetylneuraminyl residues per molecule of enzyme. When the highly purified polyisoprenyl phosphate phosphatase was analyzed by SDS-polyacrylamide gel electrophoresis, a major 33-kDa polypeptide was observed. Enzymatic dephosphorylation of Dol-P by the purified phosphatase was 1) optimal at pH 7; 2) potently inhibited by F-, orthovanadate, and Zn2+ > Co2+ > Mn2+ but unaffected by Mg2+; 3) exhibited an approximate Km for C95-Dol-P of 45 microM; and 4) was sensitive to N-ethylmaleimide, phenylglyoxal, and diethylpyrocarbonate. The pig brain phosphatase did not dephosphorylate glucose 6-phosphate, mannose 6-phosphate, 5'-AMP, or p-nitrophenylphosphate, but it dephosphorylated dioleoyl-phosphatidic acid at initial rates similar to those determined for Dol-P. Based on the virtually identical sensitivity of Dol-P and phosphatidic acid dephosphorylation by the highly purified enzyme to N-ethylmaleimide, F-, phenylglyoxal, and diethylpyrocarbonate, both substrates appear to be hydrolyzed by a single enzyme with an apparent dual specificity. This is the first report of the purification of a neutral Dol-P phosphatase from mammalian tissues. Although the enzyme is Mg2+-independent and capable of dephosphorylating Dol-P and PA, several enzymological properties distinguish this lipid phosphomonoesterase from PAP2.
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PMID:Purification and characterization of a polyisoprenyl phosphate phosphatase from pig brain. Possible dual specificity. 956 3

Purified membrane-bound alkaline phosphatase from rat osseous plate hydrolyzed pyrophosphate in the presence of magnesium ions, with a specific activity of 92.7 U/mg. Optimal apparent pH for pyrophosphatase activity was 8.0 and it remained unchanged on increasing the pyrophosphate concentration. In the absence of magnesium ions the enzyme had a Km = 88 microM and V = 36.7 U/mg for pyrophosphate and no inhibition by excess substrate was observed. Pyrophosphatase activity was rapidly destroyed at temperatures above 40 degrees C, but magnesium ions apparently protected the enzyme against denaturation. Sodium metavanadate (Ki = 1.0 mM) was a competitive inhibitor of pyrophosphatase activity, while levamisole (Ki = 8.2 mM) and theophylline (Ki = 7.4 mM) were uncompetitive inhibitors. Magnesium ions (K0.5 = 1.7 microM) stimulated pyrophosphatase activity, while cobalt (Ki = 48.5 microM) and zinc (Ki = 22.0 microM) ions were non-competitive inhibitors. Manganese and calcium ions had no effect on pyrophosphatase activity. The Mw of the pyrophosphatase protein was 130 kDa by gel filtration, but a value of 65 kDa was obtained by dissociative gel electrophoresis, suggesting that it was a dimer of apparently identical subunits. These results suggested that pyrophosphatase activity stems from the membrane-bound osseous plate alkaline phosphatase and not from a different protein.
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PMID:Inorganic pyrophosphate-phosphohydrolytic activity associated with rat osseous plate alkaline phosphatase. 959 80

Effects of static magnetic fields (SMF) on bone formation of rat femurs, were evaluated using tapered rods made of magnetized and unmagnetized samarium cobalt of the same size. They were implanted transcortically into the middle diaphysis of rat femurs under press-fit loading. The bone mineral density (BMD) and bone calcium content were measured 12 weeks after implantation by dual-energy X-ray absorptiometry and chemical analysis with o-cresolphthalein complexon, respectively. The result revealed that the femurs adjacent to magnetized specimens had significantly higher BMD and calcium content than those adjacent to the unmagnetized specimen (p < 0.01). However, the value of BMD and calcium content of rats with magnetized specimens was similar to that of non-operated rats. No specific change was found in the body weight, serum Ca, activity of alkaline phosphatase, hemogram, and BMD of the tibia and humerus among the magnetized and unmagnetized. These results suggest that the long-term local SMF stimulation on the bone has a local effect to prevent the decrease in BMD caused by surgical invasion or implantation.
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PMID:Effects of static magnetic field on bone formation of rat femurs. 979 45

The effect of standard orthopaedic materials on proliferation and differentiation of osteoblasts was examined using a standardised cell culture system. Osteoblasts hFOB 1.19 were cultured on stainless steel (SS), a chromium-cobalt-molybdenum alloy (CrCoMb) and commercially pure titanium (cpTi) for 12 days. Cell culture polystyrene (PS) was used as a reference. Cell numbers and cell viability were used as parameters of proliferation. Cell differentiation was assessed using alkaline phosphatase activity, collagen I and osteocalcin production. The parameters of proliferation showed earlier maximum values on PS and cpTi, while proliferation was delayed on SS and CrCoMb. The highest values of differentiation were found on cpTi. The development of alkaline phosphatase activity showed two peaks reflecting apoptosis and redifferentiation. The cell culture system hFOB 1.19 is thus suitable for revealing differences in proliferation and differentiation of osteoblasts on standard orthopaedic materials. The results correlate with previous in vivo findings. Using this system, the dynamic effect of the material surface on the differentiation process of osteoblasts can be demonstrated.
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PMID:[Standardized tests of bone implant surfaces with an osteoblast cell culture system. I. Orthopedic standard materials]. 1003


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