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)

A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response.
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PMID:Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity. 761 54

NADPH diaphorase is a histochemical activity which, in formaldehyde-fixed tissue, is rather specific for nitric oxide synthase. Recently, it was shown that NADPH diaphorase activity is inhibited by ethylenediaminetetraacetic acid (EDTA) in neurons but not in the choroid plexus epithelium. The present study, while confirming these results, demonstrates that the apparent sensitivity of NADPH diaphorase for EDTA reflects only the dependence of malic enzyme, which is used as the source of reduced cofactor, on Mg2+ or Mn2+ ions. Furthermore, evidence is provided that the apparent EDTA-insensitive NADPH diaphorase activity in the choroid plexus reflects the activity of alkaline phosphatase in conjunction with NADH diaphorase. Apart from these pitfalls, the use of the indirect, malic enzyme based method for NADPH diaphorase was found to cause much higher background staining compared to the direct method using NADPH, and is therefore proposed to be abandoned.
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PMID:NADPH diaphorase is not inhibited by ethylenediaminetetraacetic acid and is not specific for nitric oxide synthase in the choroid plexus of rat and mouse. 773 45

IgG antibodies against the 2,3-di-o-acyltrehalose glycolipid of Mycobacterium tuberculosis were determined in a set of 49 sera from patients with pulmonary tuberculosis and 65 negative control subjects. We compared a conventional ELISA method using a beta-galactosidase anti-human IgG conjugate developed with ONPG, with an amplification ELISA system constituted of an anti-human IgG biotinylated conjugate, a streptavidin-alkaline phosphatase complex, and NADP as a substrate. The resulting NAD was measured by using a redox enzymatic recycling system of alcohol dehydrogenase, diaphorase and iodonitrotetrazolium as chromogen. With specificity set at 92.31% in both methods, we obtained a sensitivity of 42.86% in the conventional method and a sensitivity of 61.22% in the amplified method. We conclude that by using a more sensitive method we can detect cases that otherwise could be identified as false negatives.
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PMID:Development of an enzyme-linked immunosorbent assay (ELISA) combined with a streptavidin-biotin and enzyme amplification method to detect anti-2,3-di-o-acyltrehalose (DAT) antibodies in patients with tuberculosis. 782 39

Topological structure of quinoprotein glucose dehydrogenase in the inner membrane of Escherichia coli was determined by constructing protein fusions with alkaline phosphatase or beta-galactosidase. Analysis of the fusions revealed that the dehydrogenase possesses five membrane-spanning segments, and the N-terminal and C-terminal portions resided at the cytoplasmic and periplasmic side of the membrane, respectively. These results agreed with the hydropathy profile based on its primary structure. The topological structure suggests that the predicted binding site of the prosthetic group pyrroloquinoline quinone is located at the periplasmic side and that the amino acid residues corresponding to those that were presumed to interact with ubiquinone in one subunit of mitochondrial NADH dehydrogenase also occur at the periplasmic side. When the purified glucose dehydrogenase and cytochrome o ubiquinol oxidase were reconstituted together with ubiquinone into liposomes, a membrane potential could be generated by the electron transfer at the site of the ubiquinol oxidase but not of the dehydrogenase. These results suggest that glucose dehydrogenase has a ubiquinone reacting site close to the periplasmic side of the membrane, and thus its electron transfer to ubiquinone appears to be incapable of forming a proton electrochemical gradient across the inner membrane of E. coli.
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PMID:Topological analysis of quinoprotein glucose dehydrogenase in Escherichia coli and its ubiquinone-binding site. 850 15

Human breast (MCF-7, HBL 100, T47D, BT20, HS578T), colon (HT29, CACO2, SW620, SW480, COLO320DM) and small cell lung cancer (NCI-N417, OH3, SW2) cell lines were transplanted subcutaneously into severe combined immunodeficient (SCID) mice. When sizeable tumours developed, the mice were sacrificed and the following enzyme activities were detected histochemically: presumed nitric oxide synthase-associated diaphorase (NOSaD), beta-D-glucuronidase (beta-Gluc) and non-specific alkaline phosphatase (alP). Except for HT29 and MCF-7 presumed NOSaD activity was not detected in the tumour itself or in the neo-vasculature of the tumours. beta-Gluc activity was found in all tumour cells (except N417 and COLO 320), in the necrotic parts of the tumours and in stromal cells of the tumour bed. AlP activity was present in all tumours including their necrotic areas. However, the activities of beta-Gluc and alP varied considerably even within one tumour, ranging from very weak to very strong. Principally the results show that the human/SCID mouse tumour model is well suited to test modern applications of tumour therapy involving the enzymes NOSaD, beta-Gluc and alP. In particular, antibody directed enzyme prodrug therapy concepts and activation of prodrugs by enzymes released from tumour cells into the necrotic areas of the tumour can be evaluated in this in vivo model.
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PMID:Histochemistry of therapeutically relevant enzymes in human tumours transplanted into severe combined immunodeficient (SCID) mice: nitric oxide synthase-associated diaphorase, beta-D-glucuronidase and non-specific alkaline phosphatase. 896 Mar 2

Among kidney tubular epithelial cell types, proximal tubule cells are one of the major renal targets for xenobiotics. Several in vitro culture models have been proposed for use of proximal tubule cells for in vitro pharmacotoxicology studies. This paper reports a comparative study of the response to cephaloridine exposure of two established cell lines from pig (LLC-PK1) and rabbit (LLC-RK1) kidneys and primary cultures of rat and rabbit proximal tubule cells. These cultured cells were first compared for their levels of activity of alpha-methylglucopyranoside transport, alkaline phosphatase, succinate dehydrogenase, and NADPH cytochrome c reductase, their glutathione-dependent activity levels, and their adenylate cyclase response pattern to stimulation by PTH and AVP. The results presented show major phenotypic differences between these four cellular models. The differences observed in glutathione-dependent mechanism activities and regulation may in part be responsible for the variability of the responses of these four cellular models when exposed to cephaloridine.
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PMID:Comparative impact of cephaloridine on glutathione and related enzymes in LLC-PK1, LLC-RK1, and primary cultures of rat and rabbit proximal tubule cells. 903 21

Cellular redox status and membrane protein activities were analyzed in kidneys from rats with ischemic acute renal failure (ARF). ARF was induced by clamping the left renal artery for 50 min. A parallel group of control animals was processed. In the ischemic group urea plasma levels were statistically increased as compared with the control group. Studies employing whole kidney homogenates revealed that ischemia produces an increment in lipid peroxidation levels and a reduction in glutathione concentration and in superoxide dismutase and glutathione peroxidase activities. Since lipid peroxidation may alter the function of membrane proteins we determined succinate cytochrome c reductase (SuccR), sodium-potassium ATPase (Na-K-ATPase), glucose-6-phosphatase (G-6-Pase) and alkaline phosphatase (ALP) activities in whole renal homogenates. Only G-6-Pase and ALP activities were modified by ischemia. Since ALP is a brush border membrane (BBM) enzyme and BBM is one of the main target structures in ARF, we assessed some parameters of BBM functionality. ALP, gamma-glutamyl transferase (gamma-GT) and 5'-nucleotidase (5'-NT) showed diminished activities in BBM from ischemic kidneys. Ischemia also modified the Vmax of paraaminohippuric acid (PAH) uptake without altering Km. An increment of lipid peroxidation and membrane fluidity in BBM was observed after the treatment. Total membrane proteins and protein recoveries in BBM were similar in both experimental groups. Sialic acid and sulfhydryl levels were similar in BBM from ischemic kidney and control ones. In summary, ARF induced by renal artery clamping for 50 min takes place with a significant increase in urea plasma levels. A decrease in the antioxidant defense system is detected. This induces lipid peroxidation in whole renal tissue, which may justify the diminished activities of some membrane enzymes such as G-6-Pase and ALP. A specific analysis of BBM function reveals a significant increment of lipid peroxidation which may be the cause of an increased membrane fluidity. This latter parameter might be, at least in part, responsible for the damaged function of apical ALP, 5'-NT, gamma-GT and PAH carrier.
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PMID:Impairment of cellular redox status and membrane protein activities in kidneys from rats with ischemic acute renal failure. 968 97

By using histological morphometric stereological and quantitative enzyme histochemical methods, structural and metabolic changes in thyroid gland after short-term treatment with high doses of cyclophosphamide (GY) were studied. Mice were treated with 400 mg/kg of GY every 48 h for up to 7 days(1-4 i.p. injections). To assess the effect of CY and its reversibility thyroids were studied on the day following each injection and 5-15 days after three injections. CY treatment caused significant dose-dependent structural and metabolic changes in thyroid gland which included vacuolization and destruction of thyrocyte apical cytoplasm, focal follicular wall destruction leading to the fusion of some follicles, reduction in volume fractions of epithelium with the condominant increase in volume fraction of colloid and stroma, decrease in follicular cell height, decline in both thyrocyte cytoplasmic NAD-H-diaphorase activity and vascular alkaline phosphatase activity. These changes were not completely reversible by day 15 after the last injection of CY.
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PMID:[Morphofunctional changes in the thyroid gland after cyclophosphamide administration and their reversibility]. 1158 50

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a novel nucleotide derived from NADP that has now been shown to be active in releasing Ca(2+) from intracellular stores in a wide variety of cells ranging from plant to human. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, no assay has been reported for NAADP to date. In the present study, a widely applicable assay for NAADP with high sensitivity is described. NAADP was first dephosphorylated to nicotinic acid-adenine dinucleotide by treatment with alkaline phosphatase. The conversion was shown to be stoichiometric. NMN-adenylyltransferase was then used to convert nicotinic acid-adenine dinucleotide into NAD in the presence of high concentrations of NMN. The resultant NAD was amplified by a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD cycled through these coupled reactions, a molecule of highly fluorescent resorufin was generated. The reaction could be performed for hours, resulting in more than a 1000-fold amplification. Concentrations of NAADP over the 10-20 nM range could be routinely measured. This novel cycling assay was combined with an enzymic treatment to provide the necessary specificity for the assay. NAADP was found to be resistant to NADase and apyrase. Pretreatment of samples with a combination of the hydrolytic enzymes completely eliminated the interference from common nucleotides. The versatility of the cycling assay can also be extended to measure nicotinic acid, which is a substrate in the synthesis of NAADP catalysed by ADP-ribosyl cyclase, over the micromolar range. All the necessary reagents for the cycling assay are widely available and it can be performed using a multi-well fluorescence plate reader, providing a high-throughput method. This is the first assay reported for NAADP and nicotinic acid, which should be valuable in elucidating the messenger functions of NAADP.
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PMID:A novel cycling assay for nicotinic acid-adenine dinucleotide phosphate with nanomolar sensitivity. 1211 13

THE ALDEHYDES INTRODUCED IN THIS PAPER AND THE MORE APPROPRIATE CONCENTRATIONS FOR THEIR GENERAL USE AS FIXATIVES ARE: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4 degrees C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that-notable in the case of glutaraldehyde-was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.
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PMID:Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. 1397 66


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