Alkaline phosphatase, £. coli

Angelini, S., Moreno, R., Gouffi, K. et al. (2001) Export of Thermus thermophilus alkaline phosphatase via the twin-arginine translocation pathway in Escherichia coli. FEBS Letters, 506 (2), 103-107. 

Cambella and Antia [385] determined phosphonates in seawater by fractionation of the total phosphorus. The seawater sample was divided into two aliquots. The first was analysed for total phosphorus by the nitrate oxidation method capable of breaking down phosphonates, phosphate esters, nucleotides, and polyphosphates. The second aliquot was added to a suspension of bacterial (Escherichia coli) alkaline phosphatase enzyme, incubated for 2h at 37 °C and subjected to hot acid hydrolysis for 1 h. The resultant hot acid-enzyme sample was assayed for molybdate reactive phosphate which was estimated as the sum of enzyme hydrolysable phosphate and acid hydrolysable... 

While having three metal ions in an enzyme active site is uncommon, it is not unique to PLCBc. The well-known alkaline phosphatase from E. coli (APase) contains two zinc ions and a magnesium ion [67], whereas the a-toxin from Clostridiumperfringens [68]. and the PI nuclease from Penicillium citrinum [69] each contain three zinc ions. Indeed, the zinc ions and coordinating ligands of PI nuclease bear an uncanny resemblance to those of PLCBc as the only differ-... 

Enzyme labels are usually coupled to secondary antibodies or to (strept)avidin. The latter is used for detection of biotinylated primary or secondary antibodies in ABC methods (see Sect. 6.2.1). Enzyme labels routinely used in immunohisto-chemistry are horseradish peroxidase (HRP) and calf intestinal alkaline phosphatase (AP). Glucose oxidase from Aspergillus niger and E. coli (3-galactosidase are only rarely applied. 

Enzymatic markers used in immunohistochemistry Horseradish peroxidase (HRP) and calf intestinal or E.coli alkaline phosphatase (AP). Glucose oxidase from Aspergillus niger and E.coli /3-galactosidase are only rarely applied. 

The long-lived phosphorescence of the tryptophan in alkaline phosphatase is unusual. Horie and Vanderkooi examined whether its phosphorescence could be detected in E. coli strains which are rich in alkaline phosphatase.(89) They observed phosphorescence at 20°C with a lifetime of 1.3 s, which is comparable to the lifetime of purified alkaline phosphatase (1.4 s). Long-lived luminescence was not observed from strains deficient in alkaline phosphatase. The temperature dependence of tryptophan phosphorescence in the living cells was slightly different from that for the purified enzyme, indicating an environmental effect. 

T. Horie and J. M. Vanderkooi, Phosphorescence of alkaline phosphatase of E. coli in vitro and in situ, Biochim. Biophys. Acta 670, 290-290 (1981). 

Isolation of alkaline phosphatase from Escherichia coli in which 85% of the proline residues were replaced by 3,4-dehydro-proline affected the heat lability and ultraviolet spectrum of the protein but the important criteria of catalytic function such as the and were unaltered (12). Massive replacement of methionine by selenomethionine in the 0-galactosidase of E. coli also failed to influence the catalytic activity. Canavanine facilely replaced arginine in the alkaline phosphatase of this bacterium at least 13 and perhaps 20 to 22 arginyl residues were substituted. This replacement by canavanine caused subunit accumulation since the altered subunits did not dimerize to yield the active enzyme (21). Nevertheless, these workers stated "There was also formed, however, a significant amount of enzymatically active protein in which most arginine residues had been replaced by canavanine." An earlier study in which either 7-azatryptophan or tryptazan replaced tryptophan resulted in active protein comparable to the native enzyme (14). 

The X-ray crystal structure of the inorganic phosphate (an inhibitor) complex of alkaline phosphatase from E. coli (9) showed that the active center consists of a Zn2Mg(or Zn) assembly, where the two zinc(II) atoms are 3.94 A apart and bridged by the bidentate phosphate (which suggests a phosphomonoester substrate potentially interacting with two zinc(II), as depicted in Fig. 2), and the Mg (or the third Zn) is linked to one atom of the zinc pair by an aspartate residue at a distance... 

Izutani, K., Nakata, A., Shinagawa, H. Kawamata, J. (1980) Forward mutation assay for screening carcinogens by alkaline phosphatase constitutive mutations in Escherichia coli K-12. BikenJ.,13,69-75... 

Considerable ingenuity was required in both the synthesis of these chiral compounds695 697 and the stereochemical analysis of the products formed from them by enzymes.698 700 In one experiment the phospho group was transferred from chiral phenyl phosphate to a diol acceptor using E. coli alkaline phosphatase as a catalyst (Eq. 12-36). In this reaction transfer of the phospho group occurred without inversion. The chirality of the product was determined as follows. It was cyclized by a nonenzymatic in-line displacement to give equimolar ratios of three isomeric cyclic diesters. These were methylated with diazomethane to a mixture of three pairs of diastereoisomers triesters. These dia-stereoisomers were separated and the chirality was determined by a sophisticated mass spectrometric analysis.692 A simpler analysis employs 31P NMR spectroscopy and is illustrated in Fig. 12-22. Since alkaline phosphatase is relatively nonspecific, most phosphate esters produced by the action of phosphotransferases can have their phospho groups transferred without inversion to 1,2-propanediol and the chirality can be determined by this method. 

The alkaline phosphatases are found in bacteria, fungi, and higher animals but not in higher plants. In E. coli alkaline phosphatase is concentrated in the peri-plasmic space. In animals it is found in the brush border of kidney cells, in cells of the intestinal mucosa, and in the osteocytes and osteoblasts of bone. It is almost absent from red blood cells, muscle, and other tissues which are not involved extensively in transport of nutrients. 

The alkaline phosphatase of E. coli is a dimer of 449-residue subunits which requires Zn2+, is allo-sterically activated by Mg2+, and has a pH optimum above 8.667/708 711 At a pH of 4, incubation of the enzyme with inorganic phosphate leads to formation of a phosphoenzyme. Using 32P-labeled phosphate, it was established that the phosphate becomes attached in ester linkages to serine 102. The same active site sequence Asp-Ser-Ala is found in mammalian alkaline phosphatases. These results, as well as the stereochemical arguments given in Section 2, suggest a double-displacement mechanism of Eq. 12-38 ... 

Figure 12-23 Schematic drawing of the product inorganic phosphate bound in the active site of E. coli alkaline phosphatase. See Ma and Kantrowitz.719...

Fig. 1. Specific identification off. coli containing plasmid pBR322. Approximately 225 colonics, consisting of a 10 1 mixture of plasmid-frec and plasmid-containing cells, was grown on a nitrocellulose filter. The filterwas subjected to the lysis protocol described here, followed by a hybridization with biotinylated pBR322. Sites of positive hybridization were detected by means of streptavidin and alkaline phosphatase. The dark sites correspond to colonies harboring pBR322. Plasmid-free cells give the faint signals present at numerous sites on the filter.

Alkaline phosphatase from E. coli is an enzyme of the 1960 s. Although one brief reference to a phosphatase from E. coli having an alkaline pH maximum was reported in 1933 (1), it was not until the discovery by... 

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). 

Although the enzyme sediments with intact cells, alkaline phosphatase appears in the supernate when broken cells are centrifuged. Malamy and Horecker (5) discovered that alkaline phosphatase is quantitatively released from the cell when E. coli are converted to spheroplasts by lysozyme and ethylenediaminetetraacetic acid (EDTA) in a sucrose medium. This evidence, supported by the observation that substrates such as glucose 6-phosphate are rapidly hydrolyzed by intact cells with release of most of the phosphate into the medium, led Malamy and Horecker (6) to suggest that alkaline phosphatase is localized in the periplasmic space, a region described by Mitchell (7) as lying between the protoplasmic membrane and the wall layer, and that it is not in association with the wall (8). 

Later, it was found that alkaline phosphatase was also released by osmotic shock E. coli were exposed to 0.5 M sucrose containing dilute tris-HCl buffer and EDTA, and then the centrifuged cells were rapidly dispersed in the shock medium of cold water or cold 5 X 10 4 M MgCl2. Although the cells were 80% viable with the latter case, almost all of the enzyme was released (9, 10). Other evidence indicates that the only important structural effect of EDTA is to increase the permeability of the cell wall (11, 12). Escherichia coli grow normally in the... 

Localization of the enzyme in the periplasmic space is also consistent with the selective release of alkaline phosphatase during growth of an E. coli mutant which is osmotically sensitive because of a defective cell wall (14) and with the fact that phosphate esters which do not penetrate the protoplasmic membrane can be hydrolyzed by intact cells 15). In these latter measurements the activities found with intact cells as compared with equivalent cell extracts varied over wide limits depending upon the substrate and its concentration. This difference was assumed to result from a difference in the ease of penetration of the wall barrier by different phosphate esters. 

One possibility is that the concentration of monomers in the endoplasm is too low to produce dimers and that the monomers are concentrated in the periplasmic space by active transport. Evidence against active transport was obtained in episomal transfer of the structural gene from E. coli to S. typhimurium (26). Even though S. typhimurium does not synthesize alkaline phosphatase, the enzyme was produced by the heterogenote and appeared in the periplasmic space. Schlesinger and Olsen (26) argued that it is unlikely that S. typhimurium would have a transport system for alkaline phosphatase monomers because it does not normally make the enzyme. 

Although it is widely found in bacteria, the physiological function of alkaline phosphatase is still unknown. The enzyme is nonspecific (4, 28), and this would be desirable if its role were to supply phosphate from phosphate esters under conditions of phosphate deprivation. Although the enzyme is repressed by orthophosphate in many strains of E. coli, it is constitutive in most other bacteria (29), thus phosphate deprivation... 

The methods (and procedures) for growing E. coli and purifying alkaline phosphatase have been extensively reviewed by Torriani (32, 33) (see also Table I). 

Early methods of purifying alkaline phosphatase from E. coli involved heat shock (4), disruption of cells with a French press (4, 34, 35), and... 

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