Enzyme alkaline phosphatase

Deming and Pardue studied the kinetics for the hydrolysis of p-nitrophenyl phosphate by the enzyme alkaline phosphatase. The progress of the reaction was monitored by measuring the absorbance due to p-nitrophenol, which is one of the products of the reaction. A plot of the rate of the reaction (with units of pmol mL s ) versus the volume, V, (in milliliters) of a serum calibration standard containing the enzyme yielded a straight line with the following equation... 

Alkaline phosphatase catalyzes the dephosphorylation of a mmber of artificial substrates ( ) including 3-glycerophosphate, phenylphosphate, p-nitrophenylphosphate, thymolphthalein phosphate, and phenolphthalein phosphate. In addition, as shown recently for bacterial and human enzymes, alkaline phosphatase simultaneously catalyzes the transphosphorylation of a suitable substance which accepts the phosphoryl radical, thereby preventing the accumulation of phosphate in the reaction mediim (25). 

Enzyme alkaline phosphatase Antibody anti-goat IgG 0 Antigen goat IgG... 

The on-bead assay was conducted according to Scheme 3.19, which shows the chain of events, which leads to a colorimetric response, when an oligosaccharide binds effectively to the B. purpurea lectin. The lectin was covalently linked to biotin, a small molecule with an extremely high affinity for streptavidin. The bead-lectin-biotin conjugates were then exposed to streptavidin, linked to the enzyme alkaline phosphatase. Alkaline phosphatase hydrolyses phosphate esters [e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 110]. When the 5-bromo-4-chloro-3-hydroxyindole (111) is released, in the presence of nitro blue tetrazolium (NBT), it forms a dark purple, insoluble dye, thus staining beads where there was a favorable binding interaction. 

Chapman and Breslow synthesized zinc(II) complexes of monomer and dimers derived from 1,4,7-triazacyclododecane with phenyl 48 and 4,4 -biphenyl linkers 49 (55). They were examined as catalysts for the hydrolysis of 4-nitrophenyl phosphate (NP2 ) and bis(4-nitrophenyl) phosphate (BNP ) in 20% (v/v) DMSO at 55°C. On the basis of the comparison of the pseudo-first-order rate constants, the dinuclear zinc(II) complexes 48 with 1,3-phenyl and 1,4-phenyl linkers are ca. 5 times more efficient than monomer or 49 in the hydrolysis of NP2, leading to the conclusion that the two zinc(II) ions are simultaneously involved in the hydrolysis, as in the enzyme alkaline phosphatase. For the hydrolysis of BNP, a longer dimer 49 is ca. six times more effective than 1,3-phenyl-linked dimer 48 and monomers. 

An example of an optical enzyme sensor (Arnold, 1985) in a bifurcated optical fiber is shown in Fig. 9.32. The bifurcated fiber delivers and collects light to and from the site of the enzymatic reaction. The enzyme, alkaline phosphatase (AP), catalyzes hydrolysis of p-nitrophenyl phosphate to p-nitrophenoxide ion which is being detected (A = 404 nm). 

The GC mode can also be used to image some enzymes that are not oxidoreductases. For the important enzymes alkaline phosphatase (ALP) and galactosidase, this has been achieved by using an enzyme substrate that is not redox active at the UME potential, whereas one of the products (p-aminophenol (PAP)) can be oxidized. The experiment is detailed in the Protocol P2 H37. The use of potentiometric probes is also possible. Table 37.2 provides an overview about the investigated enzymes. 

On the other hand, the main types of immunoassays that can be performed by using labelled antibodies or antigens are direct sandwich, competitive and indirect assays. The labels can be enzymes (alkaline phosphatase, peroxidise or glucose oxidase) metal NPs (gold) fluorescent or electrochemiluminescent probes. 

Serum biochemistry methods were used to evaluate the toxic effect of the procedure. The parameters evaluated include concentrations of major ions (Na+,K+,C1 ) major proteins (albumin and total protein) liver-specific enzymes (alkaline phosphatase ALP) aspartate aminotransferase (AST), alanine aminotransferase (ALT)... 

Psilocybin is activated in the body by first being converted to psilocin by the enzyme alkaline phosphatase. Psilocin is then metabolized and inactivated by monoamine oxidase to form 4-hydroxyindole-3-acetic acid. This is then excreted in the urine. 

This method was developed by Collaborative Research Inc. (F3). The technique, based on the use of immunoreactive liposomes, may be classified as another type of homogeneous immunoassay. The liposomes are microscopic vesicles (200-1000 nm in diameter) consisting of a relatively impermeable lipid bilayer that delineates and separates an internal aqueous compartment from the external aqueous medium. The principle is as follows (Fig. 3 and Table 6). An enzyme, alkaline phosphatase, is encapsulated in the liposomes [E] and sequestered from the substrate, p-nitrophenyl phos-... 

Another enzyme that was studied extensively in microreactors to determine kinetic parameters is the model enzyme alkaline phosphatase. Many reports have appeared that differ mainly on the types of enzyme immobilization, such as on glass [413], PDMS [393], beads [414] and in hydrogels [415]. Kerby et al. [414], for example, evaluated the difference between mass-transfer effects and reduced effidendes of the immobilized enzyme in a packed bead glass microreactor. In the absence of mass-transfer resistance, the Michaelis-Menten kinetic parameters were shown to be flow-independent and could be appropriately predicted using low substrate conversion data. 

Catalyst 17 is effective only with substrates that can bind to the metal ion, so we attached it - coordinated as its Ni2+ derivative - to the secondary face of a-cyclodextrin in catalyst 21 [102]. This was then able to use the metallo-oxime catalysis of our previous study, but with substrates that are not metal ligands, simply those that bind hy-drophobically into the cyclodextrin cavity. As hoped, we saw a significant rate increase in the hydrolysis of p-nitrophenyl acetate, well beyond that for hydrolysis without the catalyst or for simple acetyl transfer to the cyclodextrin itself. Since there was full catalytic turnover, we called compound 21 an artificial enzyme - apparently the first use of this term in the literature. The mechanism is related to that proposed earlier for the enzyme alkaline phosphatase [103]. 

The major psychedelic agent in psilocybian mushrooms is psilocybin— the first indole derivative discovered to contain phosphorus. When ingested, the phosphorus radical is immediately "dephosphorylated by an intestinal enzyme, alkaline phosphatase, into psilocin and phosphoric acid Animal experiments suggest that psilocybin and psilocin appear at similar chemical concentrations at about the same time in various organs. Thus, the phosphorus radical is generally considered "dead weight in terms of psychoactivity. 

The third example is a phosphoryl transfer enzyme, alkaline phosphatase. The active site of alkaline phosphatase contains two Zn + ions, with a separation of 3.9 A. One zinc center is used to bind the phosphate monoester substrate, the other to activate Ser-102 for nucleophilic attack on the phosphate group of the substrate via an associative mechanism, as shown... 

We describe simple procedures for attaching biotin to antigens, antibodies, and the enzyme alkaline phosphatase. 

Immunocytochemistry The presence of the labeled probe bound to the in situ nucleic acid target is detected by means that relate specifically to the nature of the label. The most frequently used labels are enzymes (alkaline phosphatase, hydrogen peroxidase) and fluorochromes (fluorescein, rhodamine, hydroxycoumarin). 

In this paper we report the assaying of individual molecules of the enzyme alkaline phosphatase (EC 3.1.3.1) by capillary electrophoresis (CE) utilizing laser-... 

The biologically active R- or 5 -pantothenic acid can be obtained upon hydrolysis of coenzyme A with a combiaation of two enzymes, alkaline phosphatase and pantotheiaase (13) (Fig. 1). The phosphatase catalyzes the selective cleavage of the phosphate bond ia coenzyme A to afford adenosin-3 5 -diphosphate (6) and 4-phosphopantetheiae (7). The latter substance is dephosphorylated enzymatically to yield pantetheiae (8), which is rapidly converted by pantotheiaase to pantothenic acid (1). Table 1 Hsts some physical properties of pantothenic acid and its derivatives. 

Another chemiluminescent enzyme system is based on the use of stabilized dioxetane substrates. Dio-xetanes are intermediates in many chemiluminescent reactions. It s possible to synthesize stabilized dio-xetanes (phosphatase and p-galactose moieties) that do not spontaneously react. When exposed to the right enzyme (alkaline phosphatase and p-galactosidase, respectively) the dioxetane will be destabilized and spontaneously undergo a chemiluminescence reaction. " ... 

The activities of many zinc-dependent enzymes have been shown to be affected adversely in zinc-deficient tissues. Three enzymes, alkaline phosphatase, carboxypeptidase, and thymidine kinase, appear to be most sensitive to zinc restriction in that their activities are affected adversely within three to six days of institution of a zinc-deficient diet to experimental animals. 

A new assay has recently been reported for the enzyme alkaline phosphatase. The reactions upon which the assay is based are shown below ... 

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