Alkaline phosphatase substrate specificity

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... 

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. 

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). 

A different application of visible microscopy was pioneered by Gomori. In 1941 he showed that alkaline phosphatase could be specifically located by its hydrolysis of soluble phosphate esters (initially glycerophosphate). If calcium ions were present in the medium in which the sections were incubated, insoluble calcium phosphate precipitated as a result of the action of the hydrolase. The site of the precipitate could be visualized if cobalt or lead salts were subsequently added to replace calcium and the sections exposed to hydrogen sulfide. In principle many hydrolases and other enzymes could be studied using the appropriate substrates and precipitants. It was important to ensure that the products of the enzyme reactions did not diffuse from the sites where the enzymes were located. It was also essential that the reagents could reach the enzyme site. 

Phosphatases are numerous and important enzymes (see also Chapt. 2). They are classified as phosphoric monoester hydrolases (phosphatases, EC 3.1.3), phosphoric diester hydrolases (phosphodiesterases, EC 3.1.4), triphosphoric monoester hydrolases (EC 3.1.5), diphosphoric monoester hydrolases (pyrophosphatases, EC 3.1.7), and phosphoric triester hydrolases (EC 3.1.8) [21] [63]. Most of these enzymes have a narrow substrate specificity restricted to endogenous compounds. However, some of these enzymes are active toward xenobiotic organophosphorus compounds, e.g., alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2), aryldialkylphosphatase (para-oxonase (PON1), EC 3.1.8.1) and diisopropyl-fluorophosphatase (tabunase, somanase, EC 3.1.8.2) [64 - 70]. However, such a classification is far from definitive and will evolve with further biochemical findings. Thus, a good correlation has been found in human blood samples between somanase and sarinase activities on the one hand, and paraoxonase (PON1) type Q isozyme concentrations on the other [71]. 

This enzyme [EC 3.1.3.1], also known as alkaline phos-phomonoesterase, phosphomonoesterase, and glycero-phosphatase, catalyzes the hydrolysis of many ortho-phosphoric monoesters (the substrate specificity is quite wide) to generate an alcohol and orthophosphate. The... 

The frequent occurrence of sialylated enzymes, or even of multiple forms, which are sometimes tissue-dependent, with a varying number of sialyl residues as, for example, in y-glutamyltranspeptida.se (EC 2.3.2.2),456,457 is not yet fully understood. Although the activity of most of these enzymes is not influenced by removal of sialic acid,454 the activity of monoamine oxidase A (EC 1.4.3.4) of outer mitochondrial membranes of rat liver has been shown to be destroyed by treatment with sialidase438 the substrate specificity of acetylcholinesterase (EC 3.1.1.7) is altered,459 the kinetic properties of human acid and alkaline phosphatases (EC 3.1.3.1 and 3.1.3.2) are changed, and the stability of a-D-galactosidase (EC 3.2.1.22) is drastically lowered.415 In these cases, an influence of sialyl residues on the conformation of the enzyme is assumed, but awaits firm evidence. 

The absorbance measurement conducted in these assays can be replaced by fluorescence detection (fluorescence ELISA). In this case, the enzymatic conjugate chosen is an alkaline phosphatase that converts the specific substrate into a fluorescent compound. 

By analysis of the products with glucose oxidase, it was shown that the anomeric composition of the glucose liberated from glucose-6-phos-phate by the enzymes acid or alkaline phosphatase or by glucose-6-phosphatase from rat liver was essentially the same as that of the substrate, thus indicating a lack of anomeric specificity for these enzymes also (106). 

Although the HRP/TMB system is usually a good, reliable, and sensitive combination, HRP has a number of alternative substrates, which can be used such as o-phenylene diamine. There are also number of options for the enzyme used other than HRP, such as alkaline phosphatase, which can be used in combination with the substrate p-nitrophenyl phosphate. It is important to note that if alkaline phosphatase is used, the wash buffer must not contain phosphate. Usually in this case a Tris-buffered rather than phosphate-buffered wash buffer is used. The choice of enzyme-substrate system depends on a number of factors, including price, sensitivity and whether a spectrophotometer filter is available for the substrate specific wavelength to be measured. 

Protection of the phenolic moiety as a phosphate provides substrates such as 58 that are specifically recognized and cleaved by alkaline phosphatase to afford 59 (Scheme 13) <1998PCA5406>. This type of dioxetane is behind many of the commercial assays that are discussed further in Section 2.16.10.1. 

This enzyme is a relatively substrate-specific, Mg2+-dependent, alkaline phosphatase (Parthasarathy et al., 1994), which commonly accompanies MIPS during... 

Krstulovic and co-workers (K30) reported on the development of assays for serum acid and alkaline phosphatase enzymes. These enzymes have been reported to be elevated in various disease states (A2, B14, F2, Kl). In addition, they developed a method for tryptophanase analysis using RPLC (K29). Employing fluorometric detection, the substrate, tryptophan, and reaction product, indole, can be monitored selectively with high sensitivity. They reported on several advantages of this method, including minimal sample preparation, rapid analysis time, and high specificity. 

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