Phosphatase bacterial alkaline

This section focuses on two most widely used APases, a bacterial alkaline phosphatase (BAP) from E. coli and a mammalian APase from calf intestinal mucosa (CIAP), to illustrate various strategies common to the application of APases. For the sake of general information on the diversity of mammalian APases, a brief description is given for human APases. 

BAP is encoded by the phoA gene and is synthesized as a precursor carrying an N-terminal signal sequence which is processed upon secretion into the periplasmic space. 

The reaction can be terminated in various ways. If complete elimination of BAP is not necessary, the reaction can be stopped by adding either Pj to 0.1 M final concentration or 0.1% (w/v) SDS plus 10 mM EGTA. To stop the reaction and remove BAP, usually phenol/chloroform extraction is performed twice, and the DNA is precipitated with ethanol. 

The rate and efficiency of APase reactions are dependent on various factors, especially pH, ionic strength, and temperature. 

Vanadate is a strong inhibitor of BAP with a K, of 2—3 pM (13). Phenol enhances vanadate inhibition in the hydrolysis of PNPP, presumably by forming a phenyl vanadate ester [K- = 0.2 pM) which has a greater affinity for the enzyme than the vanadate alone. Note that phenol by itself is a weak competitive inhibitor with a /Cj of 180 mM. For other metal ions, inhibition decreases in the order of tungstate (WO , Kj = 6 pM) arsenate (AsO , = 3—20 pM) molybdate 

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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... 

Alkaliphilic Bacillus sp. AM-001 was aerobically grown to the early stationary phase at 37°C in alkaline medium (pH 9.0) containing 1% konjak mannan. Total chromosomal DNA obtained by the method of Saito and Miura( 14) was digested with Hindlll restriction enzyme. And 2 to 4 kbp DNA fraction of chromosomal DNA was collected by 1% agarose gel electrophoresis. The plasmid pUC19 was digested with Hindlll and then dephosphorylated with bacterial alkaline phosphatase. After the... 

Hall (1964) used whole snake venom and bacterial alkaline phosphatase to prepare nucleosides from mixed sRNA in a search for minor bases (Hall 1965). 

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. 

Metal ions also participate in the functioning of other nucleases, although the structural details of their participation are not nearly as established as those for staphylococcal nuclease. DNAse I also requires Ca for its catalytic activity.SI endonuclease, mung bean nuclease, and Physarum polycephalem nuclease require zinc ion either as cofactors or intrinsically for nuclease activity, and the restriction enzyme EcoRI may also require intrinsically bound zinc ion. In terms of how the zinc ion might function in these enzymes, one can look both to staphylococcal nuclease and to bacterial alkaline phosphatase for some... 

Bacterial alkaline phosphatase is the gene product of phoA, a member of the pho regu-lon (Table 9.1). When the pho regulon is induced by low external quantities of phosphate, synthesis of this alkaline phosphatase can represent as much as 6 mole% of total protein synthesis, and enzyme activity per cell can increase 1000-fold (Coleman and Gettins, 1983). The enzyme is synthesized as 43,000 Da monomers, which are transported to the periplasmic space and become active only after dimerization. As with many alkaline phosphatases, this enzyme accepts a broad range of substrates, which it hydrolyses at similar rates (Fernley and Walker, 1967 Reid and Wilson, 1971). Substrates are compounds with the general formula... 

Several independent techniques have been used to identify bacterial alkaline phosphatase activity in natural bacterial assemblages. Phosphatase activity perse has often been detected using para-nitrophenyl phosphate as a substrate enzyme activity is expressed as rate of formation of the hydrolytic product, para-nitrophenol, which is easily detected spectrophotometrically because it absorbs light strongly at = 395 10 nm at pH > 9 (see Huber and Kidby, 1984, for a review of variations on this theme). Other substrates that have been used to detect alkaline phosphatase activity... 

A reversed phase procedure offering good sensitivity, selectivity and precision for the determination of all six deoxyribonucleosides with a mild sample preparation method has been reported (Kuo et al., 1980). The DNA samples were degraded to their component deoxyribonucleosides by complete digestion with the enzymes DNAase 1, nuclease PI and bacterial alkaline phosphatase. Complete separation of the six deoxyribonucleosides was achieved in 70 min on a jaBondapak Cjg reversed phase support using dual wavelength UV detection. 

Release of the nucleoside components from tRNA can be carried out by enzymatic hydrolysis with nuclease PI and bacterial alkaline phosphatase (Gehrke et al., 1982). Quantitative hydrolysis can be achieved in two hours and does not suffer from problems associated with the chemical lability of certain nucleosides (Randerath et al., 1972). A similar analysis of the nucleosides in Salmonella typhimurium tRNA hydrolysates has been reported (Buck et al., 1983). 

Brizzard, B. L., Chubet, R. G., and Vizard, D. L. (1994). Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. Biotechniques 16, 730-735. 

In a second procedure, poly(ADP-ribose) was first separated from the bulk of the nucleic acids and proteins by dihydroxyboryl-Sepharose affinity chromatography 147,190). The isolated polymer was treated with snake venom phosphodiesterase and bacterial alkaline phosphatase to yield the nucleoside 2, l"-ribosyladenosine from internal residues. This product was then treated with chloroacetaldehyde to produce the fluorescent derivative, l,iSr -ethenoribosyladenosine, which was then separated from other derivatized residues by reversed-phase high performance liquid chromatography picomole amounts were quantified by fluorescence detection. This procedure facilitates the accurate determination of minute quantities of endogenous poly(ADP-ribose) (102, 190). Niedergang et al. (147) have also utilized a fluorimetric assay for determination of the enzymatic digestion products of the polymer, ADP-ribose, or iso-ADP-ribose. 

Synthesis and purification of 2 dNAD+. We used p-NMN+ adenyl transferase to synthesize [adenylate- P]2 dNAD+ from [a-3 ]2 dATP. Residual 2 dATP was removed by affinity chromatography on a boronate gel as described elsewhere (14). The elimination of contaminating P-NMN+ (Fig. lA), which also boimd to the boronate gel, was facilitated by its conversion to nicotinamide ribose by treatment with bacterial alkaline phosphatase (Fig. IB). Figure 1C shows that following preparative HPLC, a single peak corresponding to pure 2 dNAD was detected. It is important to note that all of the 32p radiolabel co-eluated with this peak. 

Cloning of the "140 bp DNA" in M13mp8 vector. DNA from the "140 bp DNA" was subjected to SI nuclease treatment followed by a dephosphorylation using bacterial alkaline phosphatase. The DNA was en treated with T4 polynucleotide kinase and the ends repaired with T4 DNA polymerase. After each enzymatic step, the solutions were extracted with phenol and chloroform and the DNA recovered by ethanol precipitation. The blunt-ended DNA was cloned by blunt-end ligation in the Hindi site of M13mp8 vector. All these steps were achieved according to standard procedures (15). Ml 3 clones (26 total) were obtained. 

The fractions containing cross-linked species are pooled and lyophi-lized several times to remove the volatile salt. The residue is dissolved in approximately 100 1 of 10 mM ammonium carbonate at pH 7.5 and mixed with unlabeled tRNA. This mixture is then digested for 4-5 hr at 37° with Tl ribonuclease (about 5 units per Aseo of total tRNA ) and bacterial alkaline phosphatase (about 5 per A ea of total tRNA). After digestion, the reaction mixture is extracted with an equal volume of... 

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