Affinity-chromatography purification of alkaline phosphatase from calf intestine

Brenna, Oreste V.; Perrella, Michele; Pace, Mario; Pietta, Piergiorgio

doi:10.1042/bj1510291

Cited by 51 publications

(21 citation statements)

References 27 publications

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“…5). Bacillus flexus ALP presents K m values which are identical to the ALPs reported by von Tigerstrom, 1984 22 and Brenna et al, 1975 6 . One of the characteristics of nonspecific ALPs is their high sensitivity to the final end product Pi.…”

Section: Ph / Temperature Optima and Thermolability Of Alpsupporting

confidence: 74%

“…The further purification was done by affinity chromatography using phosphate analog arsenate moiety having specific affinity to ALP 6 . The arsenate is a competitive inhibitor of ALP and has been used commonly as a ligand in the affinity chromatography purification of ALP from marine Vibrio sp.…”

Section: Results and Discussion: Purification Of Alpmentioning

confidence: 99%

“…The maximum ALP activity containing eluate fraction was further purified by affinity chromatography. The stationary phase was prepared by coupling diazonium salt of 4-(paminophenylazo) phenyl arsenic acid to Sephacryl-200 HR 6 . The fraction was loaded on a Sephacryl-200 HR column (200 mm x 10 mm) preequilibrated with reaction buffer.…”

Section: Materials and Methods: Purification Of Alp From Fpb17mentioning

confidence: 99%

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Untitled

2016

IJPSR

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ABSTRACT:The extracellular alkaline phosphatase from halotolerant facultative alkaliphile Bacillus flexus FPB17 was purified through precipitation / dialysis / chromatography and resulted in 28 fold of purification. The molecular weight of this enzyme through SDS-PAGE gel is 63 ± 2 kDa. Optimum conditions for purified ALP activity were pH 9.0 and 35°C. The enzyme proved to be thermolabile and gets completely denatured at 60°C in 10 min. p-NPP was found to be a preferred substrate and Km (0.33 mM) and V max (0.2 mM mg-1) determined using p-NPP as a substrate. Inorganic phosphate proved to be a competitive inhibitor of ALP. Mg +2 and Ca +2 increased the ALP activity by 47% and 20% respectively whereas Sodium fluoride, Sodium arsenate and EDTA decreased the activity by 47%, 65% and 77% respectively. The superiority of dephosphorylation of λ phage DNA by this newly discovered alkaline phosphatase over that of calf intestinal alkaline phosphatase of M/s. HiMedia Laboratories, Mumbai opens up possibility of its future use in molecular biology as a replacement of alkaline phosphatases from Escherichia coli, calf intestine and shrimp.

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Section: Ph / Temperature Optima and Thermolability Of Alpsupporting

confidence: 74%

Section: Results and Discussion: Purification Of Alpmentioning

confidence: 99%

Section: Materials and Methods: Purification Of Alp From Fpb17mentioning

confidence: 99%

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2016

IJPSR

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“…Peak fractions were pooled and dialyzed extensively against buffer A to remove inorganic phosphate (P i ). In some instances, additional purification of the wild-type enzyme was achieved using phenyl arsenate affinity chromatography (26).…”

Section: Materials Methyl 24-dinitrophenyl Phosphate (Mdnpp)mentioning

confidence: 99%

Functional Interrelationships in the Alkaline Phosphatase Superfamily: Phosphodiesterase Activity of Escherichia coli Alkaline Phosphatase

2001

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Escherichia coli alkaline phosphatase (AP) is a proficient phosphomonoesterase with two Zn 2+ ions in its active site. Sequence homology suggests a distant evolutionary relationship between AP and alkaline phosphodiesterase/nucleotide pyrophosphatase, with conservation of the catalytic metal ions. Furthermore, many other phosphodiesterases, although not evolutionarily related, have a similar active site configuration of divalent metal ions in their active sites. These observations led us to test whether AP could also catalyze the hydrolysis of phosphate diesters. The results described herein demonstrate that AP does have phosphodiesterase activity: the phosphatase and phosphodiesterase activities copurify over several steps; inorganic phosphate, a strong competitive inhibitor of AP, inhibits the phosphodiesterase and phosphatase activities with the same inhibition constant; a point mutation that weakens phosphate binding to AP correspondingly weakens phosphate inhibition of the phosphodiesterase activity; and mutation of active site residues substantially reduces both the mono-and diesterase activities. AP accelerates the rate of phosphate diester hydrolysis by 10 11 -fold relative to the rate of the uncatalyzed reaction [(k cat / K m )/k w ]. Although this rate enhancement is substantial, it is at least 10 6 -fold less than the rate enhancement for AP-catalyzed phosphate monoester hydrolysis. Mutational analysis suggests that common active site features contribute to hydrolysis of both phosphate monoesters and phosphate diesters. However, mutation of the active site arginine to serine, R166S, decreases the monoesterase activity but not the diesterase activity, suggesting that the interaction of this arginine with the nonbridging oxygen(s) of the phosphate monoester substrate provides a substantial amount of the preferential hydrolysis of phosphate monoesters. The observation of phosphodiesterase activity extends the previous observation that AP has a low level of sulfatase activity, further establishing the functional interrelationships among the sulfatases, phosphatases, and phosphodiesterases within the evolutionarily related AP superfamily. The catalytic promiscuity of AP could have facilitated divergent evolution via gene duplication by providing a selective advantage upon which natural selection could have acted.Two divalent metal ions are a common active site feature found in a number of evolutionarily distinct classes of enzymes, including enzymes that catalyze the hydrolysis of phosphate monoesters, diesters, and triesters (for reviews, see refs 1 and 2). The structural similarity shared by these active sites raises the possibility that a single active site with two divalent metal ions might be able to catalyze these different classes of reactions. However, the partial negative charge on a phosphoryl oxygen atom of the phosphate monoester dianion is lost upon conversion to a diester monoanion, and the steric bulk of the added esterifying group of the diester could result in steric clashes with...

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“…The supernatant was applied to an affinity chromatography column (0.8 cm2 x 12 cm) equilibrated with 10 mM Tris'HCl buffer (pH 8.4). The affinity chromatography column was prepared by coupling the diazonium salt of 4-(p-aminophenylazo)phenyl arsonic acid to tyraminyl-Sepharose as described by Brenna et al (10). The column was washed with 5 mM NaCl in 10 mM Tris HCl (pH 8.4) Morita (11).]…”

mentioning

confidence: 99%

Heat-labile alkaline phosphatase from Antarctic bacteria: Rapid 5′ end-labeling of nucleic acids

Kobori

Sullivan

Shizuya

1984

Proc. Natl. Acad. Sci. U.S.A.

105

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A heat-labile alkaline phosphatase has been purified to near homogeneity from HK47, a bacterial strain isolated from Antarctic seawater. The active form of the enzyme has a molecular weight of 68,000 and is uniquely monomeric. The optimal temperature for the enzymatic activity is 250C. Complete and irreversible thermal inactivation of the enzyme occurs in 10 min at 550C. By using this heat-labile enzyme for dephosphorylation followed by a 10-min heat treatment, rapid end-labeling of nucleic acids by T4 polynucleotide kinase has been achieved.The radioactive end-labeling technique of nucleic acids by T4 polynucleotide kinase is a useful tool in studies of the structure and function of nucleic acids (1-3). The technique involves key reactions: first, the removal of the existing phosphates at the 5' termini of DNA, RNA, or oligonucleotides by alkaline phosphatase (APase), and second, the addition of the radioactive phosphates to the same 5' termini by polynucleotide kinase in the presence of ATP (4). Following the first reaction, the APase activity must be eliminated before the second reaction in order to avoid both degradation of ATP and the loss of label from the substrates. However, elimination of the APase activity following dephosphorylation by either removal or inactivation has been difficult because of the great biochemical stability of the known APase enzymes. For example, Escherichia coli APase, which is the most widely used, can be heated for 8 min at 95°C with reten-

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Affinity-chromatography purification of alkaline phosphatase from calf intestine

Cited by 51 publications

References 27 publications

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Functional Interrelationships in the Alkaline Phosphatase Superfamily: Phosphodiesterase Activity of Escherichia coli Alkaline Phosphatase

Heat-labile alkaline phosphatase from Antarctic bacteria: Rapid 5′ end-labeling of nucleic acids

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