Phosphomonoesterases alkaline

Phosphates of pharmaceutical interest are often monoesters (Sect. 9.3), and the enzymes that are able to hydrolyze them include alkaline and acid phosphatases. Alkaline phosphatase (alkaline phosphomonoesterase, EC 3.1.3.1) is a nonspecific esterase of phosphoric monoesters with an optimal pH for catalysis of ca. 8 [140], In the presence of a phosphate acceptor such as 2-aminoethanol, the enzyme also catalyzes a transphosphorylation reaction involving transfer of the phosphoryl group to the alcohol. Alkaline phosphatase is bound extracellularly to membranes and is widely distributed, in particular in the pancreas, liver, bile, placenta, and osteoplasts. Its specific functions in mammals remain poorly understood, but it seems to play an important role in modulation by osteoplasts of bone mineralization. 

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... 

Alkaline phosphomonoesterase Hydrolysis of phosphoric acid esters Index of pasteurization... 

Milk contains several phosphatases, the principal ones being alkaline and acid phosphomonoesterases, which are of technological significance, and ribonuclease, which has no known function or significance in milk. The alkaline and acid phosphomonoesterases have been studied extensively (see Andrews (1993) for references). 

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 ... 

Acid phosphomonoesterase (EC 3.1.3.2). Milk contains an acid phosphatase which has a pH optimum at 4.0 and is very heat stable (LTLT pasteurization causes only 10-20% inactivation and 30 min at 88°C is required for full inactivation). Denaturation of acid phosphatase under UHT conditions follows first-order kinetics. When heated in milk at pH 6.7, the enzyme retains significant activity following HTST pasteurization but does not survive in-bottle sterilization or UHT treatment. The enzyme is not activated by Mg2+ (as is alkaline phosphatase), but it is slightly activated by Mn2+ and is very effectively inhibited by fluoride. The level of acid phosphatase activity in milk is only about 2% that of alkaline phosphatase activity reaches a sharp maximum 5-6 days post-partum, then decreases and remains at a low level to the end of lactation. 

Diaz-Maurino, T. and Nieto, M. 1976. Milk fat globule membranes. Inhibition by sucrose of the alkaline phosphomonoesterase. Biochim. Biophys. Acta 448, 234-244. Diaz-Maurifio, T. and Nieto, M. 1977. Milk fat globule membranes Chemical composition and phosphoesterase activities during lactation. J. Dairy Res. 44, 483-493. Dowben, R. M., Brunner, J. R. and Philpott, D. E. 1967. Studies on milk fat globule membranes. Biochim. Biophys. Acta 135, 1-10. 

Horiuchi et al. (2), and Torriani (S) that orthophosphate repressed the formation of a nonspecific phosphomonoesterase in E. coli that research on this enzyme began. This work (2, 3) showed a maximum rate of synthesis of the enzyme occurred only when the phosphate concentration became low enough to limit cell growth. With sufficient phosphate, the amount of active enzyme is negligible. Under conditions of limiting phosphate, alkaline phosphatase accounts for about 6% of the total protein synthesized by the cell (4). 

Alkaline and acid phosphatase are organ-specific enzymes that are assayed in the diagnosis of many diseases. These activities are phosphomonoesterases that dephosphorylate a number of compounds, including nucleoside monophosphates, to their respective nucleosides and free phosphates. However, such dephosphorylations have traditionally been assayed with 4-nitrophenyl... 

Figure 9.93 HPLC chromatograms of phosphomonoesterase hydrolysis of A(S)MP. (i4) Chromatogram obtained from calf intestinal mucosa alkaline phosphatase hydrolysis of A(S)MP. In a reaction volume of 100 /xL containing 100 mM Tris-HCl (pH 8.1), 300 pM A(S)MP, and 20 mM MgCl2, the reaction was initiated by addition of 2 of enzyme and incubated at 30°C for 6 hours. A 20 /xL sample was then injected onto the HPLC column and analyzed. (B) Chromatogram obtained from snake venom S -nucleotidase incubated with A(S)MP. In a reaction volume of 100 /xL containing 100 mM Tris-Cl (pH 8.1), 300 jxM A(S)MP, and 20 mM MgCl2, the reaction was initiated by addition of 6 yxg of enzyme and the reaction mixture incubated at 30°C for 60 minutes, and a 20 yxL sample was injected onto the HPLC column and analyzed. (From Rossomando et al., 1983.)...

Alkaline phosphatases are typically dimeric zinc metalloenzymes ranging in size from 80 to 145 kDa. They catalyze a nonspecific phosphomonoesterase reaction of the following type ... 

Dephosphorylation may be carried out using bacterial alkaline phosphomonoesterase (Heppell et al. 1962). Typical conditions are 20 fig desalted oligonucleotide mixture in 2 ml 1 M ammonium bicarbonate with 10 fig enzyme incubated for 1 hr at 37°C (De Wachter and Piers 1967). This removes 3 -terminal phosphates. Brownlee and Sanger (1967) carried out combined T1 ribonuclease and bacterial alkaline phosphatase digestion of the low molecular weight rRNA using enzyme/substrate ratios of 1/20 and 1/5 respectively in 0.01 M Tris pH 8.0 at 37°C for 1 hr. This produces fragments like XpXp...XpG where X represents any nucleoside except G. 

The phosphomonoesterases that proved most useful in this work, although free of proteolytic impurities, were found to be complex in their behavior toward phosphate esters. As indicated in Table II, if tested with the aid of low molecular weight substrates, the intestinal (85) and the potato phosphatase (34) act on 0—P and N—P bonds, whereas the prostate enzyme (86) hydrolyzes only 0—P linkages. After the discovery of the specificity of two of these enzymes for low molecular weight N—P esters, it was noticed that the intestinal enzyme, although classified in the literature as alkaline phosphatase, hydrolyzes N—P bonds both at pH 5.6 and 9.0, but not at pH 7.0. Since the pH range of 5 to 6 is that of maximum stability of almost all proteins, most experiments were carried out in this pH range. Thus the use of these three enzymes, either alone or in combination with each other, proved to be quite a powerful tool. 

F4. Fischer, E., Effect of inhibitors on the histochemical reaction of alkaline a-naph-thyl and naphthol-AS-phosphomonoesterase in the kidney of edible-frog (Rana Esculenta L.) Acta Biol. AmmJ. Sd. Hung. 16, 383-388 (1966). 

Alkaline phosphomonoesterase. Phosphomonoesterase. An orthophosphoric monoester + H(2)0 an alcohol + phosphate. 

Alkaline phosphatases (AP EC 3.1.3.1) are dimeric, zinc-containing, non-specific phosphomonoesterases which are found in... 

This enzyme is an alkaline phosphomonoesterase that catalyzes the hydrolysis of nucleoside 5 -monophosphates (e.g., adenosine-5 -monophosphate and inosine-5 -monophosphate). It appears to be distributed widely in the body tissues and to be mainly a membrane-bound enzyme, but it is also found in the cytoplasm, lysosomes, and other subcellular organelles. In the liver, the enzyme is located on the bile canicular membrane and on other cells, including the sinusoidal cells, leading to its use for the detection of hepatobiliary injury (Goldberg 1973 Dooley and Racich 1980 Carakostas, Power, and Banerjee 1990 Sunderman 1990). The enzyme measurement has largely been discarded in clinical medicine however, more recently, the 5 -NT enzymes have been implicated in the inhibition of antiviral nucleosides, so interest may be revived (Hunsucker, Mitchell, and Spychala 2005). 

Treatment of bromomethyl acetate with the sodium salt of a dialkyl phosphite, followed by deacetylation with methoxide affords the corresponding dialkyl hydroxymethanephosphonate. If this is tosylated with tosyl chloride, and the product treated with nucleosides [protected at the 3 -(and 2 -, if present) hydroxy groups] and sodium hydride in DMF, the monoalkyl ester of a 5 -0-phosphonyl-methyl nucleoside (60) is obtained after deblocking the sugar, and subsequent dealkylation with TMS-iodide affords (61). Like alkyl esters of 5 -mononucleotides, (60) is resistant to acid and alkaline hydrolysis, while (61) is stable in acidic and alkaline media, and is also resistant to hydrolysis by alkaline phosphomono-esterase and snake venom 5 -nucleotidase. Treatment of (61) with DCC and morpholine, and subsequently with orthophosphate or pyrophosphate, affords (62) and (63), respectively. Alkaline phosphomonoesterase from E. co/i hydrolyses the pyrophosphate links in (62) and (63) to give (61) and orthophosphate. The UTP and CTP analogues (63 B = U or C) are inhibitors of uridine kinase from... 

Enzymatic reactions can be used as a means of both characterizing and releasing classes of organic phosphorus present in waters, soils and sediments. For example, phosphate monoesters can be quantified by the use of a phosphomonoesterase such as alkaline phosphatase. Alternately, the high specificity of some enzymes for particular substrates can be used as the basis for determination of specific organic phosphorus compounds, or as part of a post-separation quantification step. 

Many living cells contain at least two types of phosphomonoesterase enzymes (EC 3.1.3), the acid and the alkaline phosphatases. 

Colvan, S.R., Syers, J.K. and O Donnell, A.G. (2001) Effect of long-term fertiliser use on acid and alkaline phosphomonoesterase and phosphodiesterase activities in managed grassland. Biology and Fertility of Soils 34, 258-263. 

Most of the monometallic hydrolases contain the zinc(II) ion in their catalytic centers as an essential metal ion. Typical zinc(II) hydrolases include carboxypeptidase (CPD), thermolysin, /3-lactamase, phosphomonoesterase (such as alkaline phosphatase (AP)), phosphodiesterase, phosphotriesterase, matrix metalloproteinase (MMP), and carbonic anhydrase (CA). ... 

Depending on pH, phosphomonoesterases have been grouped into acid phosphatase and alkaline phosphatase. Optimum pH range of 4-6 for acid phosphatase and S.3-9.5 for alkaline phosphatase... 

Fig. 8. Outlines of the method to identify isomers of phosphoribosyl pentapeptide. 1-, 2-, and 3-peptidyl ribose 5-phosphate are expected to give, after oxidation by NaI04 (pH 7, room temperature, 60 min), reduction by NaBH4, and digestion with alkaline phosphomonoesterase, glycerol (C3), ribitol (C5), and erythritol (C4), respectively. For reference, the expected production of ethylene glycol (Cj), which is mostly distilled out at the step of evaporation in vacuo, from free ribose 5-phosphate is also shown...

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