Phosphatase alkaline

Alkaline phosphatases [AP, orthophosphoric-monoester phosphorylase (alkaline optimum) EC 3.1.3.1] represent a large family of almost ubiquitous isoenzymes found in organisms from bacteria to animals. In mammals there are two forms of alkaline phosphatase, one form present in a variety of tissues and another form found only in the intestines. They share common attributes in that the phosphatase activity is optimal at pH 8 to 10, is activated by the presence of divalent cations, and is inhibited by cysteines, cyanides, arsenate, various metal chelators, and phosphate ions. Most conjugates created with alkaline phosphatase utilize the form isolated from calf intestine. 

AP isoenzymes can cleave associated phosphomonoester groups from a wide variety of substrates. The exact biological function of these enzymes is still uncertain. They can behave in vivo in their classic phosphohydrolase role at alkaline pH, but at neutral pH AP isoenzymes can act as phosphotransferases. In this sense, suitable phosphate acceptor molecules can be utilized in solution to increase the reaction rates of AP on selected substrates. Typical phosphate acceptor additives include diethanolamine, Tris, and 2-amino-2-methyl-l-propanol. The presence of these additives in substrate buffers can dramatically increase the sensitivity of AP ELISA determinations, even when the substrate reaction is done in alkaline conditions. 

Purified preparations of calf intestinal AP maintained in solution are usually stored in the presence of a stabilizer, typically 3 M NaCl. The enzyme also may be lyophilized, but may experience activity loss with each freeze-thaw cycle. AP is not stable under acidic conditions. Lowering the pH of an AP solution to 4.5 reversibly inhibits the 

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can withstand the moderately high temperatures associated with hybridization assays better than HRP, AP often is the enzyme of choice for labeling oligonucleotide probes. AP also is capable of maintaining enzymatic activity for extended periods of substrate development. Increased sensitivity can be realized in ELISA procedures by extending the substrate incubation time to hours and sometimes even days. These properties make AP the second most popular choice for antibody-enzyme conjugates (behind HRP), being used in almost 20 percent of all commercial enzyme-linked assays. 

Alkaline phosphatase160-164 is a dimeric zinc metalloenzym composed of two identical subunits. The number of zinc atoms per protein molecule varies in different preparations. However, only two seem to be required for catalytic activity. The molecular weight of the monomer has been reported to be 42.000 so the natural dimer would be twice that value. Alkaline phosphatase is a phosphorylating enzyme and has 760 residues per dimer. 

Zinc can be removed165 and the resulting apoalkaline phosphatase binds no phosphate with a dissociation constant smaller than 5 x 10-5 M. Upon addition of metal ions to the apoenzyme, a high affinity binding site may be reactivated. Two metals are needed to form one highly specific phosphate binding site. 

The action of phosphatase on glucose-l-phosphate substrate is shown in Fig. 18. Phosphatase breaks the P-0 bond and releases the P04 group. In contrast phosphorylase which operates on the same substrate opens the C—0 bond. Apparently this affinity to specific bond sites, which in the present case amounts to a difference of ca. 1.5 A (Fig. 18) between the two bonds left and right from the oxygen, is 

The anion binding properties of alkaline phosphatase from Escherichia 

The metal-free apoenzyme may be readily obtained and, furthermore, 

In view of the conflicting ideas of the metal-binding of alkaline phosphatase it was of interest to attempt to elucidate this 

The same stoichiometry has also been obtained with Cd 

Hg and Mn [136]. The most obvious interpretation of these findings is that, for an enzyme containing the minimum amount of zinc needed for full activity, there is no coordination of Cl ions to the protein-bound zinc ions. That metal-coordinative halide binding is not signi- 

Variations between species using different enzyme substrates have been shown for cholinesterase (Myers 1953 Evans 1990) and other enzymes (e.g., angiotensin converting enzyme Evans 1989). For alkaline phosphatase, the majority of methods employ 4-nitrophenylphosphate as the substrate, but there are two main alternative buffers—diethanolamine and 2-aminopropanol—that can cause interspecies differences (Masson and Holmgren 1992). For isoenzymes, the majority of laboratories currently use a variety of electrophoretic separation methods although selective inhibition of isoenzymes with antibodies is being used increasingly, there are problems associated with protein specificity and relative isoenzyme concentrations in animal samples. 

ALT (also known as glutamic pyruvic transaminase, GPT) catalyzes the reaction 

In rodents, preanalytical factors such as food intake and restraint may alter plasma ALT (see Chapter 12 also). A 50% food restriction over 210 days in rats resulted in elevated ALT values compared to controls (Schwartz, Tornaben, and Boxhill 1973), while reduced levels of ALT were observed in a study of fasting effects on the oral toxicity of several xenobiotics (Kast and Nishikawa 1981). Changes of ALT related to diet may reflect perturbations of gluconeogenesis (Toropila et al. 1996). There are now several examples where plasma ALT falls after the administration of xenobiotics due to effects on pyridoxal phosphate, which is a cofactor necessary for action of the aminotransferases AST and ALT (Dhami et al. 1979 Rhodes et al. 1987 Waner et al. 1990 Waner and Nyska 1991). Such effects may confuse the interpretation of data when hepatotoxicity occurs and tends to increase ALT, but where there is an opposing effect due to reductions in pyridoxal phosphate. Further complications with ALT have been described by Wells and To (1986) in covalent binding studies with acetaminophen. 

Some investigators have expressed the activities of the two aminotransferases as a ratio (AST ALT) to assist with interpretation. However, it is necessary for each laboratory to establish its own discriminating ratios because the use of different methods and animal species affects the values obtained for these ratios. From the factors affecting these measurements in different species, it can be seen that ratios established for one species cannot be used for another species. 

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Craig D B, Arriaga E A, Wong J C Y, Lu H and Dovichi N J 1996 Studies on single alkaline phosphatase molecules reaction rate and activation energy of a reaction catalyzed by a single molecule and the effect of thermal denaturation—the death of an enzyme J. Am. Chem. See. 118 5245-53... 

Polakowski R, Craig D B, Skelley A and Dovichi N J 2000 Single molecules of highly purified bacterial alkaline phosphatase have identical activity J. Am. Chem. See. 122 4853-5... 

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... 

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). 

Phosphatase Test. The phosphatase [9001-78-9] test is a chemical method for measuring the efficiency of pasteurization. AH raw milk contains phosphatase and the thermal resistance of this enzyme is greater than that of pathogens over the range of time and temperature of heat treatments recognized for proper pasteurization. Phosphatase tests are based on the principle that alkaline phosphatase is able, under proper conditions of temperature and pH, to Hberate phenol [108-95-2] from a disodium phenyl phosphate substrate. The amount of Hberated phenol, which is proportional to the amount of enzyme present, is determined by the reaction of Hberated phenol with 2,6-dichloroquinone chloroimide and colorimetric measurement of the indophenol blue formed. Under-pasteurization as well as contamination of a properly pasteurized product with raw milk can be detected by this test. 

Pa.g et s Disease of Bone. Paget s disease, osteitis deformans, occurs mainly ia people over 40. About twice as many men as women are affected. The disease, caused by faulty utilisation of may be mild and asymptomatic requiring Httle or no treatment. Clinical signs are high alkaline phosphatase and high urine hydroxyproline as weU as abnormal bone stmcture which usually goes unrecognised until discovered accidentally by routine x-ray examination (32). 

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

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). 

For most assays, the incorporated pantothenic acid has to be Hberated en2ymatically. Usually, a combination of pantotheinase and alkaline phosphatase is used to hberate the bound pantothenic acid. The official method for pantothenic acid of the Association of Official Analytical Chemists (AOAC) is the microbiological assay that uses U. Plantarium (A.TCC 8014) as the test organism (71). Samples are extracted at 121°C at pH 5.6—5.7, proteins are precipitated at pH 4.5, and the resulting clear extracts are adjusted to pH 6.8 prior to assay. This procedure is only suitable to determine calcium pantothenate or other free forms of pantothenic acid. 

Rapid inactivation of added lincomycin was found to result from the growth of Streptomjces rochei in a synthetic medium the antibiotic was converted into lincomycin 3-phosphate [23670-99-7] (5, R = CH3, R = PO3H2, R = SCH3), C2gH33N202PS, readily cleaved back to the antibiotic upon treating with alkaline phosphatase (53). 

The dosage of flucytosine is 150—200 mg/kg orally in four portions every six hours. A 1% flucytosine solution has been developed for intravenous adrninistration. In some countries, a 10% ointment is also available. In patients with normal renal function, flucytosine is seldom toxic, but occasionally severe toxicity may be observed (leukopenia and thrombocytopenia). Plasma levels should be determined and the dose in patients with impaired renal function should be checked. Liver function tests (transaininases and alkaline phosphatase) should be performed regularly. In some patients with high flucytosine plasma levels, hepatic disorders have been observed (24). 

Aplastic anemia and leukemia are not the only health effects ascribed to benzene exposure. A number of recent studies have associated benzene exposure with chromosomal changes (aberrations) (118). Other studies have shown abnormalities in porphyrin metabolism and decrease in leucocyte alkaline phosphatase activity in apparendy healthy workers exposed to 10—20 ppm benzene (119,120). Increases in leukoagglutinins, as well as increases in blood fibrinolytic activity, have also been reported and are believed to be responsible for the persistent hemorrhages in chronic benzene poisoning (121,122). 

An enzyme-amplified detection scheme, based on tire coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the elecn oiiractive a-naphthyl phosphate to a-naphtlrol this product is elecU oactive and has been detected by means of differential... 

Alkaline phosphatase from rat osteosarcoma has been purified by acetone pptn, followed by chromatography on DEAE-cellulose, Sephacryl S-200, and hydroxylapatite. [Nair et al. Arch Biochem Biophys 254 18 1987.]... 

Enzymes are powerful catalysts. Enzyme-catalyzed reactions are typically 10 to times faster than their uncatalyzed counterparts (Table 16.1). (There is even a report of a rate acceleration of >10 for the alkaline phosphatase-catalyzed hydrolysis of methylphosphate )... 

Simoponlos, T. T, and Jencks, W. P., 1994. Alkaline phosphatase is an almost perfect enzyme. Biochemistry 33 10375-10380. 

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