Proteolytic modification of Escherichia coli alkaline phosphatase

Tyler-Cross, R; Roberts, C H; Chlebowski, J F

doi:10.1016/s0021-9258(18)83774-7

Cited by 14 publications

(4 citation statements)

References 18 publications

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“…However, it appears more likely that Mg 2+ binding causes long-range intersubunit interactions that change the overall AP structure significantly. This agrees with previous results from experiments with trypsin-modified AP hybrid dimers which revealed that the modified subunit altered the structural and kinetic properties of the other subunit through subunit interactions ( − ). NMR studies of metal hybrid AP dimers, with Zn 2+ bound to one subunit and Cd 2+ bound to the other, have found that Zn 2+ binding caused substantial changes in the chemical shift of the Cd 2+ ligands over 30 Å away at the other active site, again demonstrating long distance subunit interactions ().…”

Section: Discussionsupporting

confidence: 92%

“…The B sites bind Zn 2+ more strongly in the presence of phosphate and can also bind Mg 2+ at high concentrations (14); however, Mg 2+ normally occupies the C sites. There appears to be significant cooperativity among metal binding sites and between the two subunits (15,(17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

“…The B sites bind Zn 2+ more strongly in the presence of phosphate and can also bind Mg 2+ at high concentrations (); however, Mg 2+ normally occupies the C sites. There appears to be significant cooperativity among metal binding sites and between the two subunits ( 15 , − .

1 Ribbon diagram of the AP structure with two Zn 2+ and one Mg 2+ binding sites indicated at the monomer active site ().…”

mentioning

confidence: 99%

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Mg²⁺ Binding to Alkaline Phosphatase Correlates with Slow Changes in Protein Lability

2001

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The in vitro reactivation of unfolded Escherichia coli alkaline phosphatase (AP) in the presence of the two natively bound metals Zn2+ and Mg2+ produces two protein species, characterized by different guanidine hydrochloride denaturation kinetics. The high-lability AP form slowly converts to the low-lability form in a first-order reaction with a characteristic lifetime (inverse rate constant) of approximately 300 h at pH 8.0 and 25 degrees C. Addition of Zn2+ and Mg2+ ligands to (folded) apo-AP also produces two protein species, with denaturation kinetics and a long conversion lifetime similar to those found in refolding AP. In contrast, adding Zn2+ alone to apo-AP produces only the high-lability species with no subsequent structural change, suggesting that Mg2+ binding is the event which is responsible for the production of the low-lability AP. The rate of conversion from high- to low-lability AP was found to be linearly dependent on Mg2+ concentration, indicating that Mg2+ binding is rate limiting for this reaction. Experiments where either Zn2+ or Mg2+ was added first, with the second metal added later, show that Mg2+ binding is slowed by the prior presence of bound Zn2+. Mg2+ binding to Zn-AP also slightly increases the enzymatic activity; however, the extent of formation of the low-lability species is related to the square of the Mg2+-induced activity increase. Thus the binding of two Mg2+ to AP produces the dramatic reduction in the rate of denaturation that characterizes the low-lability species. The data suggest the possibility of long distance intersubunit interactions and a role for Mg2+ in providing "kinetic stability" for AP.

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Section: Discussionsupporting

confidence: 92%

mentioning

confidence: 99%

1 Ribbon diagram of the AP structure with two Zn 2+ and one Mg 2+ binding sites indicated at the monomer active site ().…”

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confidence: 99%

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Mg²⁺ Binding to Alkaline Phosphatase Correlates with Slow Changes in Protein Lability

2001

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“…The enzymatic activities of AP and the scFv-AP fusion protein were measured according to the method described by Tyler-Cross et al 26 The kinetics parameters were determined on the basis of utilizing p-NPP as the substrate. One unit of enzyme was dened as the quantity required for liberating 1 mM of 4-nitrophenol per h. 27 The reaction was monitored at 25 C by following the increase in absorbance at 405 nm (A405 nm) in a 1 M Tris-HCl buffer (pH ¼ 8.0) containing 10 mM MgCl 2 and 50 mM ZnCl 2 .…”

Section: Enzymatic Activity Analysismentioning

confidence: 99%

A one-step chemiluminescence immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single chain Fv–alkaline phosphatase fusion protein

Tao

Shen

et al. 2015

Anal. Methods

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A one-step generic chemiluminescence competitive direct enzyme immunoassay (CL-cdELISA) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein (CL-cdELISA scFv-AP ) was developed. It was capable of detecting 20 targeted fluoroquinolones (FQs) in fish and shrimp matrices within 40 min below the maximum residue levels (MRLs). In the optimized generic assay, the scFv-AP fusion protein in combination with a norfloxacin-ovalbumin conjugate (NOR-OVA) showed 50% binding inhibition (IC 50 ) at 0.15 AE 0.01 mg kg À1 for NOR in 0.01 M phosphate-buffered saline (PBS), indicating that it is seven times as sensitive as the corresponding competitive direct enzyme immunoassay (cdELISA scFv-AP ), and the linear response range of the assay was extended from 0.04 to 1.08 mg kg À1 . The limits of detection (LODs) of the assay for NOR were 0.017 mg kg À1 in shrimp and 0.018 mg kg À1 in fish, and the LODs inferred from the cross reactivity (CR) ranged from 0.013 mg kg À1 for ciprofloxacin (CIP) to 4.19 mg kg À1 for trovafloxacin (TRO); the recoveries of the three representative antibiotics norfloxacin, flumequine (FLU) and sarafloxacin (SAR) from spiked fish and shrimp samples varied from 72.50 to 118.50% and the mean coefficients of variation for the inter-assay and intra-assay were 6.4% and 9.2%, respectively. Further validation of CL-cdELISA scFv-AP with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that the assay was a reliable screening tool for the detection of FQs in fish and shrimp.

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Engineering subtilisin BPN′ for site‐specific proteolysis

Carter¹,

Nilsson²,

Burnier³

et al. 1989

Proteins

110

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A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN', which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wild-type subtilisin BPN' is greatly restricted by substitution of the catalytic histidine-containing of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J.A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.

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Proteolytic modification of Escherichia coli alkaline phosphatase

Cited by 14 publications

References 18 publications

Mg²⁺ Binding to Alkaline Phosphatase Correlates with Slow Changes in Protein Lability

Mg²⁺ Binding to Alkaline Phosphatase Correlates with Slow Changes in Protein Lability

A one-step chemiluminescence immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single chain Fv–alkaline phosphatase fusion protein

Engineering subtilisin BPN′ for site‐specific proteolysis

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Proteolytic modification of Escherichia coli alkaline phosphatase

Cited by 14 publications

References 18 publications

Mg2+ Binding to Alkaline Phosphatase Correlates with Slow Changes in Protein Lability

Mg2+ Binding to Alkaline Phosphatase Correlates with Slow Changes in Protein Lability

A one-step chemiluminescence immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single chain Fv–alkaline phosphatase fusion protein

Engineering subtilisin BPN′ for site‐specific proteolysis

Contact Info

Product

Resources

About

Mg²⁺ Binding to Alkaline Phosphatase Correlates with Slow Changes in Protein Lability

Mg²⁺ Binding to Alkaline Phosphatase Correlates with Slow Changes in Protein Lability