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)

Antibodies (abs) against the terminal complement complex (C5b-9) were used on routinely processed post mortem myocardial tissue in parallel with conventional staining methods. Both monoclonal and polyclonal abs were tested using the avidin biotin peroxidase complex (ABC), alkaline phosphatase anti-alkaline phosphatase (APAAP) methods and an ab-bridge with alkaline phosphatase. Enhancement of the diaminobenzene (DAB) end product with cobalt-nickel (ABC method) was also done. The polyclonal ab gave the most satisfactory results and the alkaline phosphate conjugated ab-bridge had a slight advantage over the ABC method. Cobalt-nickel enhancement of DAB improved the visualization, but with higher background staining. APAAP was the least satisfactory method. Comparing the immunohistochemical method with the conventional staining methods, the former showed positive reaction in 97% of areas of coagulation necrosis and in 65% of contraction band necrosis. On the other hand coagulation necrosis was seen in 44% and contraction band necrosis in 68% of C5b-9 positive areas indicating that C5b-9 abs react with ischemically damaged myocytes before visible alterations are seen in hematoxilin-eosin staining. Moreover, using C5b-9 abs, it seems possible to exclude agonal/artefactual contraction bands which show a negative reaction. Immunohistochemical detection of C5b-9, using an adequate technique could increase the possibility to demonstrate early ischemic myocardial damage.
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PMID:Immunohistochemical detection of early myocardial infarction. An evaluation of antibodies against the terminal complement complex (C5b-9). 749 83

Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The M(r) of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mumol min-1 mg-1),ATP (42.0 mumol min-1 mg-1) and pyrophosphate (28.4 mumol min-1 mg-1). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited "Michaelian" kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (Kd = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (Kd = 6.2 microM) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K0.5 = 10.1 microM), magnesium (K0.5 = 29.5 microM) and manganese ions (K0.5 = 5 microM) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K0.5 = 653 microM) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.
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PMID:Osseous plate alkaline phosphatase is anchored by GPI. 808 Dec 65

Freeze-substituted rat liver embedded in glycol methacrylate (GMA) has been used to demonstrate the activities of several enzymes. The following enzymes could be detected in GMA-sections by the indicated histochemical procedure(s): 5'-nucleotidase (lead salt, cerium-diaminobenzidine), alkaline phosphatase (indoxyl-tetrazolium salt), catalase (diaminobenzidine), acid phosphatase (diazonium salt), lactate dehydrogenase (tetrazolium salt) and glutamate dehydrogenase (tetrazolium salt). The activities of all these enzymes were dramatically decreased compared with the activities demonstrated in unfixed cryostat sections, with the exception of catalase. The activities of the following enzymes could not be detected in GMA-sections: glucose-6-phosphate dehydrogenase (tetrazolium salt), xanthine oxidoreductase (tetrazolium salt), D-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide) and glucose-6-phosphatase (cerium-diaminobenzidine). The possible role of restricted penetration of reagents into the resin was studied by measuring cytophotometrically the enzyme activities in GMA-sections of 3 and 6 microns in thickness. For all the enzymes that could be detected, the 6 microns:3 microns ratio varied from 1.4 to 2.7. An eventual retarded penetration of reagents into the resin was investigated by measuring cytophotometrically the amount of final reaction product during incubation for acid phosphatase and glutamate dehydrogenase activities. In both cases linear relationships without a lag phase were found for the specific enzyme activities with incubation time. Chemical denaturation of proteins or masking of active sites in proteins due to embedding in the resin monomer may be considered to be the main cause of decreased enzyme activities.
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PMID:Quantitative aspects of enzyme histochemistry on sections of freeze-substituted glycol methacrylate-embedded rat liver. 827 44

The effect of cations on the thermophilic character of alkaline phosphatase from Thermoactinomyces vulgaris, is described. The optimal pH and temperature were 9.5 and 55 degrees C to 65 degrees C, respectively. The partial removal of cations with ethylene diamine tetraacetic acid converted the enzyme to mesophilic and susceptible to chemical denaturation. Their complete removal caused complete inhibition. The addition of 0.3mM cobalt and 10mM magnesium added before heating were found to be optimal for restoring its thermophilic character and its stability to a chemical denaturant.
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PMID:The effect of cations on the thermophilic character of alkaline phosphatase from Thermoactinomyces vulgaris. 849 May 79

Liver microsomes from rats treated with various P450 inducers were examined for their ability to metabolize the mycotoxin ochratoxin A (OTA) to 4(R)-4-hydroxyochratoxin A (4R), the major metabolite, and 4(S)-4-hydroxyochratoxin A (4S), the minor metabolite. Pretreatment of rats with phenobarbital (PB), dexamethasone (DXM), 3-methylcolcanthrene (3MC) and isosafrole (ISF) greatly induced 4R formation. PB, DXM, 3MC, clofibrate (CLF) and ISF treatments also induced 4S formation. Isoniazid (INH) pretreatment primarily induced 4S formation. The pH optimum for 4R formation was found to be 6.0 with 3MC microsomes, and 6.5 with PB and DXM microsomes. For 4S formation, the pH optimum was 7.0. At the optimum pH (compared with pH 7.4), 4R formation increased 40-50% with PB and DXM microsomes but 8.0-fold with 3MC microsomes. Studies using the inhibitors metyrapone and alpha-naphthoflavone as well as monoclonal antibodies against various P450s suggested that at least the P450 isoforms IA1/IA2, IIB1 and IIIA1/IIIA2 are involved in 4R formation. Using urinary excretion of the enzymes alkaline phosphatase and gamma-glutamyl transferase as an index of renal damage, we observed that pretreatment of rats with PB, which induced hepatic P450 (P450II2B1), protected against OTA nephrotoxicity, whereas cobalt-protoporphyrin IX pretreatment, which decreased P450 levels, exacerbated OTA nephrotoxicity. Our results suggest that at least P450IIB1-dependent metabolism of OTA leads to its detoxication and that OTA itself may be toxic in some circumstances or that other pathways are responsible for its activation.
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PMID:Effect of cytochrome P450 induction on the metabolism and toxicity of ochratoxin A. 857 85

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by cobalt ions in a similar way as by magnesium ions. For concentrations up to 1 microM, the Chelex-treated enzyme was stimulated by cobalt ions, showing Kd = 6.0 microM, V = 977.5 U/mg, and site-site interactions (n = 2.5). Cobalt-enzyme was highly unstable at 37 degrees C, following a biphasic inactivation process with inactivation constants of about 0.0625 and 0.0015 min-1. Cobalt ions stimulated the enzyme synergistically in the presence of magnesium ions (Kd = 5.0 microM; V = 883.0 U/mg) or in the presence of zinc ions (Kd = 75.0 microM; V = 1102 U/mg). A steady-state kinetic model for the modulation of enzyme activity by cobalt ions is proposed.
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PMID:Mechanism of action of cobalt ions on rat osseous plate alkaline phosphatase. 858 69

The effect of essential trace metals on bone metabolism was investigated in the femoral-metaphyseal tissues obtained from skeletal-unloaded rats. Skeletal unloading was designed by using the model of hindlimb suspension in rats; the animals were fed for 4 days with the unloading. Femoral-metaphyseal tissues were cultured for 24 hours in a medium containing either vehicle (control), nickel, manganese, cobalt, copper, zinc, or zinc-chelating dipeptide (beta-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10(-6) to 10(-4) M. Bone biochemical components (alkaline phosphatase activity, glucose consumption, and DNA content) were significantly decreased by skeletal unloading. The presence of zinc sulfate or AHZ (10(-5) and 10(-4) M) caused a significant increase of alkaline phosphatase activity in the bone tissues from unloaded rats. This effect was not seen by nickel, manganese, cobalt and copper (10(-6) to 10(-4) M). The culture medium glucose was clearly consumed by the bone tissues. This consumption was inhibited by nickel, manganese, or copper (10(-5) and 10(-4) M), while cobalt, zinc, and AHZ had no effect. DNA content in the bone tissues from unloaded rats was significantly increased by all metal compounds (10(-5) M). The effect of AHZ on bone components was greater than zinc sulfate. The AHZ (10(-5) M)-increased alkaline phosphatase activity in the bone tissues from unloaded rats was clearly blocked by the presence of cycloheximide (10(-6) M), staurosporine (10(-7) M), dibucaine (10(-4) M), or okadaic acid (10(-7) M). The present study demonstrates that, of various essential trace metals, zinc compounds have an unique anabolic effect on bone metabolism in the femoral-metaphyseal tissues of rats with skeletal unloading. Zinc-chelating dipeptide may stimulate bone protein synthesis through the mechanism that is involved in protein kinases.
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PMID:Effect of essential trace metal on bone metabolism in the femoral-metaphyseal tissues of rats with skeletal unloading: comparison with zinc-chelating dipeptide. 866 81

Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.
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PMID:Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification. 875 Nov 58

Uptakes of (28)Mg at 10 s were measured at 0.1 and 1mM MgCl(2), to mainly represent one or other of the two uptake mechanisms earlier shown to be present in rat jejunal brush border membrane vesicles, one with an apparent KT of 0.2 mM, the other in the millimolar range. Both mechanisms had an optimal temperature close to 28 degrees C, inactivation at 37 degrees C being more acute for the low affinity mechanism (55 percent, P < 0.01). Both mechanisms were equally stimulated by an electrical potential difference (negative inside the vesicles) imposed by a potassium gradient and not affected by the nature of the anion accompanying magnesium. At 0.1 mM MgCl(2), the uptake was increased by an outwardly directed proton gradient, pH 8.2 outside and 7.4 inside (38 percent, P < 0.05), but not depressed when the gradient was in the opposite direction, pH 6.6 outside and 7.4 inside. It was trans-stimulated by magnesium, strongly inhibited by amiloride and to a smaller extent by furosemide, but uninfluenced by 0.1 mM NaCl or by 100 mM NaCl, NaSCN or KCl. A slight but significant inhibition (20-30 per cent) was recorded in the presence of 0.1 mM CoCl(2), NlCl(2) or BaCl(2). At 1 mM MgCl(2), the uptake was not influenced by pH gradient, was not trans-stimulated by Mg and was not affected by furosemide. A 40 percent inhibition by amiloride was, however, recorded. Also 100 mM NaCl or KCl doubled the uptake, while 1 mM NaCl or 100 mM NaSCN did not affect it. In contrast, all the divalent cations tested produced an inhibition (from 60 to 12 percent) in the following order: Co > or = Mn > Ca > or = Ni> Ba > Sr, when used at the same concentration as magnesium. The results showed that cobalt and calcium were not true competitors. In conclusion, two distinct mechanisms would operate magnesium entry at the brush border: (1) an electrogenic high affinity Mg/Mg,H exchange, sensitive to amiloride and furosemide, and (2) an electrogenic low affinity mechanism, inhibited by the presence of several divalent cations and dependent on the presence or activity of alkaline phosphatase.
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PMID:Characterization of two mechanisms of (28)Mg uptake in rat jejunal brush border membrane vesicles. 886 Nov 33

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is known to induce orthotopic and ectopic bone formation in vivo. Several in vitro studies using rat or mouse clonal cell lines have shown that rhBMP-2 may be involved in the differentiation of osteoblasts from osteoblast precursor cells or stromal cells in the bone marrow. However, there is little information available about the effects of rhBMP-2 on cultured human bone marrow cells. We investigated the effects of rhBMP-2 cultured on human bone marrow cells and osteoblastic cells on various biomaterials. Human bone cells were divided into fresh bone marrow cells, fibroblast colony-forming units (cfu-F, stromal precursors), and osteoblastic cells. The cells were cultured with or without rhBMP-2 on various biomaterials, including titanium alloy, pure titanium, cobalt alloy, and hydroxyapatite. It was found that rhBMP-2 (500 ng/mL) significantly stimulated alkaline phosphatase production by fresh bone marrow cells and cfu-F. However, when cultured on titanium alloy or pure titanium, only fresh bone marrow cells showed an increase of alkaline phosphatase production after rhBMP-2 stimulation. Production of osteocalcin, a marker of mature osteoblasts, was not stimulated by rhBMP-2 in any combinations tested. These findings suggest that rhBMP-2 may be involved in inducing the differentiation of osteoblast precursor cells into osteoblastic cells rather than stimulating further differentiation of osteoblastic cells into mature osteoblasts. In addition, grafts of fresh human bone marrow cells of cfu-F stimulated by rhBMP-2 may have the potential to promote bone formation at sites of nonunion as well as around titanium joint prostheses.
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PMID:Effects of recombinant human bone morphogenetic protein-2 on human bone marrow cells cultured with various biomaterials. 913 62


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