Glucose 6-phosphate alkaline phosphatase

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Testicular effects were also investigated after oral administration of 2000 mg/kg bw di(2-ethylhexyl) phthalate for seven consecutive days to 13-week-old male Wistar rats (Saxena et al., 1985). Degeneration was observed in about 40% of the seminiferous tubules. Loss of succinic dehydrogenase, NADH-diaphorase and acid phosphatase activity and increases in adenosine triphosphatase, glucose-6-phosphate dehydrogenase and alkaline phosphatase activity were observed in treated rats. 

Although the enzyme sediments with intact cells, alkaline phosphatase appears in the supernate when broken cells are centrifuged. Malamy and Horecker (5) discovered that alkaline phosphatase is quantitatively released from the cell when E. coli are converted to spheroplasts by lysozyme and ethylenediaminetetraacetic acid (EDTA) in a sucrose medium. This evidence, supported by the observation that substrates such as glucose 6-phosphate are rapidly hydrolyzed by intact cells with release of most of the phosphate into the medium, led Malamy and Horecker (6) to suggest that alkaline phosphatase is localized in the periplasmic space, a region described by Mitchell (7) as lying between the protoplasmic membrane and the wall layer, and that it is not in association with the wall (8). 

Agren 112) and Engstrom 113) isolated serine phosphate from mammalian alkaline phosphatase that had been incubated with inorganic phosphate in acid pH (<6). Engstrom 114) and Schwartz and Lipmann 35) later obtained similar results with E. coli alkaline phosphatase. They found that a large percentage of the enzyme is phosphorylated, that compounds like glucose 6-phosphate and sodium arsenate inhibit... 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

This feature has been extensively investigated by Engstrom (20, 71, 88, 168, 169 see also Sections I,A and II,B) whose results may be summarized as follows (1) incubation of alkaline phosphatase with 32P-Pi at pH 4—6 and 0°, followed by acid inactivation, leads to the appearance of the label in the enzyme protein (2) after acid hydrolysis the only labeled amino acid found is phosphoserine (3) one mole of Pi is incorporated per mole of enzyme (4) the presence of Zn2+ in the enzyme is essential for phosphorylation (5) bound Pi can be displaced by addition of glucose 6-phosphate to the phosphorylation medium and (6) very little phosphoryl enzyme is formed under alkaline conditions. 

Phosphatase Hydrolysis of phosphate Translocation of ATPases linked to cation Inorganic pyrophosphatase (pyrophosphate orthophosphate + P043-) Glucose-6-phosphatase Fructose-1,6-bisphosphatase Alkaline phosphatase Phosphoprotein phosphatase (Na+, K+)-ATPase (Mg2+, Ca2+)-ATPase Proton-translocating ATPase Zn2+ Zn2+ Sodium pump Calcium pump H+ pump... 

Dixit, P. K. Quantitative histochemistry of cartilage. Alkaline phosphatase and glucose-6-phosphate dehydrogenase activity in different zones of rachitic rat cartilage during healing. Calc. Tiss. Res. JO, 49-57 (1972). 

Methods Adapted. At the present time, six methods of interest to clinical chemists are available for analysis with the Autoanalyzer. They are methods for glucose, urea nitrogen, chloride, calcium, inorganic phosphate, and alkaline phosphatase. Micromodifications are available... 

Common chromogen systems currently in use include diaminobenzidine (DAB), 3-amino-9-ethyl-carbazole (AEC), Hanker-Yates reagent, alpha-naphthol pyronin used with peroxidase as substrate fast blue, fast red, BCIP- (5-bromo-4-chloro-3-indolyl phosphate) NBT (nitroblue tetrazolium) used with alkaline phosphatase as substrate tetrazolium, tetranitroblue tetrazolium used with glucose oxidase as substrate, and immunogold with silver enhancement (Leong, 1993 Leong et al, 1997a). 

Sucrose phosphate synthetase catalyzes the reaction of UDP-glucose with fructose-6-P to form sucrose-6-P and UDP. This step is the penultimate step in the synthesis of sucrose in leaves. The chromatographic method can be applied to many UDP-glucose-requiring enzymes. The method eliminates the need for treatment of reaction mixtures with alkaline phosphatase. 

Luminescent endpoints are available for all of the commonly used enzyme labels, including horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, glucose oxidase, and P-galactosidase. Detection limits in the subattomolar range have been attained (34). Chemiluminescent assays for novel enzyme labels such as xanthine oxidase have also been developed (50). 

In the enzyme-multiplied immunoassay techniques (EMIT), the antigen or antibody is labeled with an enzyme (e.g., iysozyme, alkaline phosphatase, horseradish peroxidase, or glucose-6-phosphate dehydrogenase) instead of a radioisotope. For example, an alkaline phosphatase-labeled drug can be made to compete with an unlabeled drug for binding sites on... 

The direct detection of electrochemical labels entails problems with sensitivity. For this reason the majority of electrochemical immimoassay development has focused on the measurement of enzyme labels by detection of eiectroactive products arising from enzyme catalyzed reactions. A wide variety of enzyme labels have been used for electrochemical immunoassays. These include glucose oxidase, glucose-6-phosphate dehydrogenase and alkaline phosphatase. ... 

Foscarnet competitively inhibits Na -Pj cotransport in animal and human kidney proximal tubule brush border membrane vesicles, reversibly inhibiting sodium-dependent phosphate transport [48, 49]. Renal cortical Na-K-ATPase and alkaline phosphatase activity are not inhibited by foscarnet, nor is proline, glucose, succinate, or Na" transport [48,49]. Foscarnet induces isolated phosphaturia without hypophosphatemia in thyroparathyroidectomized rats maintained on a low phosphorus diet, without affecting glomerular filtration rate, urinary adenosine 3 5 -cyclic monophosphate (cAMP) activity, or urinary calcium, sodium or potassium excretion [48,50]. Sodium-Pj cotransport in brush border membrane vesicles from human renal cortex was reported to be even more sensitive to inhibition by foscarnet than in rat renal brush border membrane vesicles [49]. 

Phosphates have been detected through several catalytic cascades using the inhibition of alkaline phosphatase and coupling glucose oxidase [405] or... 

Alternatively, glucose-6-phosphate can be determined by using a sequence of alkaline phosphatase (EC 3.1.3.1) and GOD. Such a sequence electrode has been used for the measurement of the competitive inhibitor of phosphatase, inorganic phosphate (see Section 4.4). 

ADP AFP ab as ALAT AP ASAT ATP BQ BSA CEH CK CME COD con A CV d D E E EC ECME EDTA EIA /e FAD FET FIA G GOD G6P-DH HBg HCG adenosine diphosphate a-fetoprotein antibody antigen alanine aminotranferase alkaline phosphatase aspartate aminotransferase adenosine triphosphate benzoquinone bovine serum albumin cholesterol ester hydrolase creatine kinase chemically modified electrode cholesterol oxidase concanavalin A coefficient of variation (relative standard deviation) layer thickness diffusion coefficient enzyme potential Enzyme Classification enzyme-chemically modified electrode ethylene diamine tetraacetic acid enzyme immunoassay enzyme loading factor flavin adenine dinucleotide field effect transistor flow injection analysis amplification factor glucose oxidase glucose-6-phosphate dehydrogenase hepatitis B surface antigen human chorionic gonadotropin... 

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