Alkaline phosphatase specificity

Primary cultured porcine or bovine brain capillary endothelial cells have been used as an in vitro model for the BBB. Recently, an immortalized cell line has been established from mouse, rat, and human brain capillary endothelial cells by infection with Simian virus 40 or transfection of SV40 large T antigen (45 -7). Tatsuta et al. established an immortalized mouse brain capillary endothelial cell line (MBEC4). The activity of y-glutamyl transpeptidase and alkaline phosphatase, specific marker enzymes for brain capillary endothelial cells, was half that in the brain capillary (45). Also, P-gp was expressed on the apical membrane of MBEC4 cells, which corresponds to the abluminal membrane of the brain... 

Sadovsky, E., and Zuckerman, H., An alkaline phosphatase specific to normal pregnancy. Obstet. Gynecol. 26, 211-214 (1965). 

Interestingly, all these compounds are inactive against mouse lAPs, therefore, unsuitable for studies of lAP function in mouse models. The objectives of the current studies were the identification of small-molecule modulators of mouse lAP and, potentially, identification of novel selective scaffolds of human lAP. To this end, we applied and optimized the chemiluminescent assays that were successfully utilized for screening human lAP, TNAP, and FLAP and developed a novel assay for mouse lAP, analogous to the assays of other isozymes. These assays utilize CDP-star, a substrate of alkaline phosphatases specifically invented for and commonly utilized in blotting techniques [19, 20]. Development and utilization of the prototype plate-reader enzymatic assay for TNAP isozyme with CDP-star substrate was previously described in detail elsewhere [21]. 

Gianinazzi-Pearson V, Gianinazzi S (1978) Enzymatic studies on the metabolism of vesicular arbuscular mycorrhiza n. Soluble alkaline phosphatase specific to mycorrhizal infection in onion roots. Physiol Plant Pathol 12 45-53... 

Phosphorothioates generally protect normal tissues more than tumors. Tumor protection reported in some animal studies can pardy be explained by physiological effects of the particular dmgs, which are specific to rodents (4). WR-2721 does not appear to protect human and most animal tumors, apparentiy because of the low availabiUty of the dmg to tumor cells (4). Many tumors appear to have a reduced capillary density (44), which may mean that these tumors have altered levels of alkaline phosphatase, the enzyme that converts WR-2721 to WR-1065. A reduced abiUty of thiols to protect the hypoxic cells characteristic of many tumors may also contribute to their selectivity for normal tissues. The observation that WR-1065 protects cultured normal human fibroblasts, but not fibrosarcoma tumor cells, suggests that additional factors may contribute to the selectivity of radioprotection by WR-2721 m vivo (18). 

Alkaline phosphatase assays based on 3-glycerophosphate now appears to be obsolete, and methods buffered by either glycine or barbital are also obsolete as these buffers inhibit ALP or are poor buffers. Serum alkaline phosphatase is known to be composed of several isoenzymes which presumably arise from bone, liver, intestine, and placenta. The placental alkaline phosphatase is known to be much more resistant to heat denaturation than the other isoenzymes, and this resistance provides a simple test for it (5). The other enzymes can be separated through the differential inhibition by phenylalanine, by electrophoresis and by specific antibodies. However, the clinical usefulness of the results obtained is in doubt (23). 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

Rengefors, K., Ruttenberg, K.C., Haupert, L.C., Taylor, C., Howes, B.L. and Anderson, D.M. (2003). Experimental investigation of taxon-specific response of alkaline phosphatase activity in natural freshwater phytoplankton. Limnolology and Oceanography 48 1167-1175. 

Figure 13.8 The etoposide-alkaline phosphatase ADEPT system. Refer to text for specific details...

A measurement of renal function (creatinine and/or BUN) is an essential test for most clinical studies, as is the inclusion of an panel of liver function tests (SGOT, SGPT, LDH, CPK, GGT, and/or alkaline phosphatase). The specific tests chosen to be included in a study are somewhat dependent on both the investigator s and/or clinical scientist s experiences and the characteristics of the drug. Other important parameters to measure include serum electrolytes and at least some of the tests listed in Table 20.12. 

Several zinc enzymes that catalyse the hydrolysis of phosphoesters have catalytic sites, which contain three metal ions in close proximity (3-7 A from each other). These include (Figure 12.11) alkaline phosphatase, phospholipase C and nuclease PI. In phospholipase C and nuclease PI, which hydrolyse phosphatidylcholine and single-stranded RNA (or DNA), respectively, all three metal ions are Zn2+. However, the third Zn2+ ion is not directly associated with the dizinc unit. In phospholipase C, the Zn-Zn distance in the dizinc centre is 3.3 A, whereas the third Zn is 4.7 and 6.0 A from the other two Zn2+ ions. All three Zn2+ ions are penta-coordinate. Alkaline phosphatase, which is a non-specific phos-phomonoesterase, shows structural similarity to phospholipase C and PI nuclease however,... 

The RPIA technology has been enhanced in the Stratus CS system by utilization of a dendrimer-antibody complex in which the analyte-specific capture antibody is covalenty coupled onto a dendrimer. The test packs in the Stratus CS system include dendrimer-capture antibody complex reagent, the alkaline phosphatase labeled antibody conjugate reagent, the substrate-wash reagent and a piece of glass fiber filter paper as the solid phase. Preparation and unique properties associated with these dendrimer-coupled antibody complexes are described below. 

Theory Alkaline phosphatase is responsible for the cleavage of O-P bonds. It is found to be relatively non-specific and this characteristic permits the AP level to be assayed based on the fact that/i-nitrophcnylphosphate ion gets converted top-nitrophenolate anion at pH 10.5 as expressed in the following reaction. 

Part—I has three chapters that exclusively deal with General Aspects of pharmaceutical analysis. Chapter 1 focuses on the pharmaceutical chemicals and their respective purity and management. Critical information with regard to description of the finished product, sampling procedures, bioavailability, identification tests, physical constants and miscellaneous characteristics, such as ash values, loss on drying, clarity and color of solution, specific tests, limit tests of metallic and non-metallic impurities, limits of moisture content, volatile and non-volatile matter and lastly residue on ignition have also been dealt with. Each section provides adequate procedural details supported by ample typical examples from the Official Compendia. Chapter 2 embraces the theory and technique of quantitative analysis with specific emphasis on volumetric analysis, volumetric apparatus, their specifications, standardization and utility. It also includes biomedical analytical chemistry, colorimetric assays, theory and assay of biochemicals, such as urea, bilirubin, cholesterol and enzymatic assays, such as alkaline phosphatase, lactate dehydrogenase, salient features of radioimmunoassay and automated methods of chemical analysis. Chapter 3 provides special emphasis on errors in pharmaceutical analysis and their statistical validation. The first aspect is related to errors in pharmaceutical analysis and embodies classification of errors, accuracy, precision and makes... 

In this method the single-stranded DNA fragment to be sequenced is end-labelled by treatment with alkaline phosphatase to remove the 5 phosphate, followed by reaction with 32P-labelled ATP in the presence of polynucleotide kinase, which attaches 32P to the 5 terminal. The labelled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base as follows. 

An elegant approach is to capture the target DNA or RNA with specific oligonucleotides on to a microwell plate. Synthetic branched DNA bearing multiple alkaline phosphatase-labeled probes hybridizes to the target. A chemiluminescent substrate is added to produce signal. This branched DNA assay has been used in infectious disease detection (W3). 

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