Alkaline phosphatase, modification

Alkaline phosphatase, modification of amperometric biosensor based on carbon fiber microelectrodes,... 

Numerous modifications of in vitro culture systems have been developed for the estimation of BBB transfer [52]. Culture systems in use are either primary cultures of brain microvessel endothelial cells (BMEC) or immortalized endothelial cell hues. BMEC may be grown in co-culture with astrocytes or in astrocyte-conditioned medium. Astrocyte-derived factors increase the tightness of the barrier as measured by transendothelial electrical resistance (TEER) and by the permeability of hydrophUic markers such as sucrose. They also up-regulate the expression of BBB-enriched enzymes such as y-glutamyl transpeptidase (y-GTP) and alkaline phosphatase. A setup of the in vitro technique in a transwell system for transport studies is depicted in Figure 2.5. 

GTPCH (EC 3.5.4.16) converts the substrate GTP to 7,8-dihydroneopterin triphosphate (H2NTP) and formate. GTPCH activity is determined by measuring neopterin, the completely oxidized and dephosphorylated H TP-product of the enzyme reaction. Conversion of H2NTP to neopterin is carried out after the enzymatic reaction in presence of iodine at pH 1.0, followed by dephosphorylation with alkaline phosphatase at pH 8.5-9.0. Neopterin is detected fluorimetrically at 350/440 nm upon HPLC separation. The assay is based with some modifications on the methods published by Viveros et al. and Hatakeyama and Yoneyama [15,16]. 

Alkaline phosphomonoesterase (EC 3.1.3.1). The existence of a phosphatase in milk was first recognized in 1925. Subsequently characterized as an alkaline phosphatase, it became significant when it was shown that the time-temperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those required to destroy Mycobacterium tuberculosis, then the target micro-organism for pasteurization. The enzyme is readily assayed, and a test procedure based on alkaline phosphatase inactivation was developed for routine quality control of milk pasteurization. Several major modifications of the test have been developed. The usual substrates are phenyl phosphate, p-nitrophenyl-phosphate or phenolphthalein phosphate which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol or phenolphthalein, respectively ... 

Nucleic acids can also be biotinylated by nonenzymatic methods with photobiotin, a photoactivatable biotin analog (6), which can be commercially obtained from BRL, Sigma, and other commercial sources I have not compared the suitability of this method of biotin incorporation with that reported here, but expect that the method would be fully acceptable FMC (Rockland, ME) markets an alternate nonradioactive sequence detection kit known as Chemiprobe. The basis of this system is a chemical modification of cytosine residues m the probe DNA. After hybridization, the probe is detected by means of a monoclonal antibody that specifically recognizes the sulfonated DNA. Detection of the bound monoclonal antibody is achieved by means of an alkaline phosphatase-conjugated second antibody. 

Alkaline phosphatase is an enzyme of the cellular membranes. Its isoforms can be found in liver, digestive tract, placenta, and some tumor tissues. Bone isoform, BALP, is a membrane enzyme of the osteoblasts. Bone, liver, and intestinal isoforms are the posttranslational modifications of the same isoenzyme exprimed by the same gene, and the difference among them lies in various ways of reaction with a saccharide component, sialic acid (M9). 

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