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)

We have developed a reliable procedure for isolating endosteal osteoblasts from mouse trabecular bone. Endosteal osteoblasts were obtained by migration and proliferation of the cells from the metaphyseal bone surface of caudal vertebrae onto nylon meshes. The isolated cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The cell population consisted of 95% alkaline-phosphatase-positive cells. The cell level of alkaline phosphatase was elevated (1.19 +/- 0.26 (SD) mumol PNP/mn/mg protein) and the enzyme activity was heat-inhibitable, indicating its skeletal origin. Light and electron microscopic observation revealed that cells have morphologic and ultrastructural appearance of typical osteoblasts with high protein synthesis activity. Osteoblasts grown in multilayers in the presence of 50 micrograms/ml ascorbic acid produced within 4 days an abundant fibrous intercellular collagenous matrix forming nodules in which osteocyte-like cells were embedded. Immunolabeling revealed synthesis of type I collagen but no detectable type III collagen. In presence of 7 mM beta-glycerophosphate the matrix became mineralized after 14-21 days of culture. Mineralization could not be induced by mouse skin fibroblasts cultured under similar conditions. The mineral deposits were closely associated with the collagen matrix, consisted of EDTA-removable, Von Kossa and alizarin red S stainable material and were composed of hydroxyapatite crystals identified by X-ray electron probe microanalysis. The isolated endosteal osteoblasts also displayed an intense (+457%) increase in intracellular cAMP production in response to human (1-34) PTH (2 x 10(-8) M) stimulation. The confluent cells responded to 20 nM 1,25(OH)2D3 by a significant 45% reduction in heat labile alkaline phosphatase activity. This procedure allowed us to isolate from trabecular bone a cell population that differentiates into osteoblasts in vitro, respond to calcitropic hormones and that retains its capacity to form a calcified bone tissue in culture. This method provided us a culture system for investigating the differentiation and metabolism of endosteal osteoblastic bone forming cells.
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PMID:Characterization of endosteal osteoblastic cells isolated from mouse caudal vertebrae. 284 14

Glucuronidation of 4-nitrophenol, nopol (a monoterpenoid alcohol) and bilirubin, which in the rat, are catalyzed by three different enzymes, has been examined in liver biopsies from patients with various liver diseases, in particular cholestasis. These different activities were not correlated, which strongly suggests that at least three independently regulated forms of UDP-glucuronosyltransferases were present in the microsomes. Non ionic detergents (Triton X100, Emulgen 911) and deoxycholate produced similar activation (more than 2-fold) of the glucuronidation of 4-nitrophenol. Amphipathic substances, such as CHAPS (3-[3-cholamidopropyl-dimethylammonio]-1-propane sulfonate), and lysophosphatidylcholines maximally increased this UDP-glucuronosyltransferase activity, the most potent being oleoyl lysophosphatidylcholine (4-fold increase). Discriminant analysis of the data revealed no correlation between the three different UDP-glucuronosyltransferase activities and the age or sex of the patients. A good correlation was found on multidimensional analysis between form 1 of the enzyme (4-nitrophenol glucuronidation) and, in decreasing order of magnitude, epoxide hydrolase (measured with benzo(a)pyrene-4,5-oxide as substrate), cytochrome P-450, 7-ethoxycoumarin deethylase, aspartate aminotransferase and gamma-glutamyltransferase (r = 0.89); and between Form 3 of the enzyme (bilirubin glucuronidation) and NADPH cytochrome c reductase, alkaline phosphatase, (r = 0.81). These relationships may reflect the differential variation in enzymatic activities in various hepato-biliary diseases.
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PMID:Properties of human hepatic UDP-glucuronosyltransferases. Relationship to other inducible enzymes in patients with cholestasis. 288 32

This rapid, reproducible method for separating and determining individual alkaline phosphatase (EC 3.1.3.1) isoenzymes in serum is based on high-performance liquid chromatography with a weak anion-exchange column (SynChropak AX 300). The isoenzymes so resolved are detected by using an on-line enzyme reaction followed by spectrophotometric monitoring at 405 nm of the 4-nitrophenol formed. Complete diagnostic profiles of the various isoenzymes present in normal and pathological sera are obtained within 20 min. The mean (and SD) normal concentrations of the bone B1 and intestinal isoenzymes in serum of adults were 3.7 (4.3) and 4.5 (3.9) U/L, respectively (n = 14), and of the bone isoenzyme B2 and liver isoenzymes L1 and L2, 5.8 (8.6), 33.0 (10.6), and 12.0 (4.8) U/L, respectively (n = 17). Concentrations of the B2 and L1 isoenzymes in adults over age 40 years differed significantly from those in adults younger than 40 years, that of bone isoenzyme being lower (P less than 0.05) and that of the liver isoenzyme being higher (P less than 0.001) in the younger adults.
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PMID:Alkaline phosphatase isoenzymes in serum determined by high-performance anion-exchange liquid chromatography with detection by enzyme reaction. 319 76

Mouse ileal alkaline phosphatase is a sialyl enzyme (12-14 moles per mole of enzyme). When partially desialylated by treatment with neuraminidase, the enzyme loses most of its activity, associated with reduced apparent Vmax and Km. Part of that loss, however, is recovered as the product 4-nitrophenol's concentration builds up in the cuvette. Experimental results are presented to demonstrate that the activation is due to the binding of 4-nitrophenol as a ligand by the partially desialylated enzyme and that both the loss of activity by sialic acid removal and activation by ligand-binding are correlated with changes in protein conformation.
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PMID:Desialylated alkaline phosphatase: activation by 4-nitrophenol. 647 97

An enzyme-linked immunosorbent assay is described for the assay of anti-DNA antibodies. The method employs plastic surface for immobilization of the antigen and alkaline phosphatase-linked rabbit anti-human IgG for the detection of immune complex using PNP-P and 4MU-P as substrates. The sensitivity of the assay increased by as much as 16-fold when fluorogenic substrate was used instead of conventional PNP-P and could therefore be employed for the detection of low avidity antibodies. Using PNP-P as substrate 57% of SLE patients were positive for DNA antibody, but if 4MU-P was introduced as substrate, 71% gave a positive response. Moreover, using a fluorogenic substrate, it was possible to minimise the amount of antigen (2 nM bp). The technique is simple, reproducible and of high sensitivity.
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PMID:Enzyme-linked immunosorbent assay for anti-DNA antibodies using fluorogenic and colorigenic substrates. 660 Nov 54

To show adenylate cyclase (AC) activity in rat calvaria, it is necessary first to decalcify the specimen. In hard tissues, several enzymes (adenosine triphosphatase (ATPase), alkaline phosphatase (APase), adenylate cyclase (AC) and perhaps pyrophosphatase (PPiase) are able to degrade adenosine triphosphate (ATP). The presence of sodium fluoride (NaF) in the incubation medium reduces the quantity of precipitate formed, compared to that observed using a NaF-free incubation medium. Levamisole, used under the same conditions, gives similar results. Possibly NaF inhibits pyrophosphohydrolase and/or phosphatases which mask the AC activity. Adenylylimidophosphate (AMP-PNP), which is a specific AC substrate, confirms the results obtained with ATP. AC activity is demonstrated cytochemically in the osteoblast and preosteoblast membranes, at the junction between two osteoblasts and along the cytoplasmic processes of the osteoblast which penetrate into the osteoid matrix. The osteocytes never show a precipitate, except those which present some osteoblastic features and then only on the membrane facing the osteogenic layer. An intracellular reaction is also evident and is discussed. Parathyroid hormone (PTH) does not reveal new sites of AC activity but increases the quantity of precipitate observed.
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PMID:An attempt at localizing adenylate cyclase in rat calvaria. Influence of sodium fluoride and parathyroid hormone. 700 93

Adenylate cyclase from rat hippocampus was separated by electrophoresis in polyacryl amide microgels and stained for enzymatic activity using a new histochemical procedure. This method involves the use of AMP-PNP, aminophylline, dithiotreitole, and Sr2+ as "primary" capture ions, thus fulfilling all the demands for a really specific histochemical incubation medium for the enzyme. The incubation of the gels with this medium resulted in the inhibition of other enzymes, which are capable of splitting AMP-PNP (ATP: pyrophosphatase, alkaline phosphatase), whereas adenylate cyclase remained highly active under these conditions. The enzyme was found to be present in two forms in the gels. Both protein bands were stimulated by the addition of various biogenic amines to the incubation medium. One protein band was fully GMP-PNP dependent in its activity. It is reasonable to suppose that these forms are either differently high aggregated molecules of the enzyme or enzyme molecules bound to their regulatory sites.
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PMID:Specific demonstration of rat brain adenylate cyclase in polyacryl amide microgels by a new histochemical procedure. 732 48

In activity determination of alkaline phosphatase (AP), measuring temperature, type and concentration of buffer, and protein concentration in the test influence the molar absorptivity of 4-nitrophenol. Thus systematic errors of up to 3% may occur in activity determinations of AP if these influences are not taken into account.
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PMID:On the influence of reaction conditions in activity determination of alkaline phosphatase on the molar absorptivity of 4-nitrophenol. 735 31

We describe specifications for high-purity 4-nitrophenol, which is suitable for spectrophotometric standardization. Such a reference material is needed in clinical enzymology to establish the proper molar absorptivity of 4-nitrophenol under final reaction conditions, particularly for measuring alkaline phosphatase activity in human serum. Some lots of 4-nitrophenol available commercially met these specifications, but several did not. The latter can be purified to meet our specifications by recrystallization or sublimation. The molar absorptivity of 4-nitrophenol (35 mumol/L) IN 10 mmol/L NaOH at 25 degrees C at 401 nm is 18380 +/- 90 L.mol-1.cm-1.
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PMID:High-purity 4-nitrophenol: purification, characterization, and specifications for use as a spectrophotometric reference material. 737 Nov 50

We studied 53 lots of 4-nitrophenyl phosphate (I), obtained from 20 different commercial suppliers, and used this information to set specifications for it. Using these well-defined specifications, we classified 21 lots of I as "unacceptable," 26 lots as "borderline," and six as "acceptable." All lots were shown to contain some 4-nitrophenol and inorganic phosphate. However, "acceptable" I had < 0.3 mmol of 4-nitrophenol and < 10 mmol of inorganic phosphate per mole of I. The mole concentration of I (based on disodium hexahydrate, formula weight 371) was determined by enzymic conversion to 4-nitrophenol in five lots of "acceptable" materials. The mole fraction of I ranged from 0.982 to 0.998. From these measurements and from estimates of impurities that absorb at 311 nm, as determined by liquid chromatography and spectrophotometry at other wavelengths, our best estimate of the molar absorptivity of I at 311 nm in 10 mmol/L NaOH at 25 degrees C was 9867 L x mol-1 x cm-1, with a total uncertainty of 76 L x mol-1 x cm-1. We recommend that I used in clinical laboratories for measurement of alkaline phosphatase activity in serum meet the specifications given in this paper: I content > 98%, maximum activity > 98% in comparative testing with other "acceptable" lots of I, and impurities not to exceed the values cited above.
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PMID:4-nitrophenyl phosphate--characterization of high-purity materials for measuring alkaline phosphatase activity in human serum. 744 94


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