Thermal and pH stabilities of alkaline phosphatase from bovine intestinal mucosa: a FTIR study

Fournière, Laurence de La; Nosjean, Olivier; Buchet, René; Roux, B.

doi:10.1016/0167-4838(95)00020-u

Cited by 41 publications

(17 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is well established that CD spectroscopy is useful to detect α-helix structures, whereas infrared spectroscopy can more easily monitor β-sheet structures. Therefore, both spectroscopic techniques can provide complementary information about secondary structure changes of proteins (58)(59)(60). Infrared spectra clearly indicated an increase of β-sheet structure at the expense of other types of secondary structures upon mild acidification.…”

Section: Discussionmentioning

confidence: 99%

Acidic pH-induced folding of annexin VI is a prerequisite for its insertion into lipid bilayers and formation of ion channels by the protein molecules

et al. 2001

View full text Add to dashboard Cite

Annexin VI (AnxVI), a member of a family of Ca 2+ -and lipid-binding proteins, can interact with membranes in a Ca 2+ -independent manner and behave as a membrane integral protein. The molecular mechanisms for such behavior of AnxVI remain, however, elusive. By determining the surface pressure of phospholipid monolayers in the presence of AnxVI in a Ca 2+ -free medium, it was observed that AnxVI can penetrate the hydrophobic region of the membrane at mildly acidic pH. A pH-dependent binding of the protein was also observed with asolectin liposomes and a pull-down assay (half maximal binding at pH 5.3). The Ca 2+ -independent, pHinduced binding of AnxVI to membranes was a prerequisite for the formation of ion channels by AnxVI, as characterized by the planar lipid bilayer method. Below pH 6.0 and in the nanomolar concentration range, AnxVI formed voltage-dependent ion channels with no specificity to the transported ions. These channels were inhibited by neither La 3+ nor Zn 2+ . The single channel conductance amounted to 24.3 pS under symmetric ionic conditions. Measurements of the enhancement of fluorescence of a hydrophobic probe, 2-(p-toluidino)naphthalene-6-sulfonic acid, in the presence of AnxVI as a function of pH revealed that AnxVI exposes hydrophobic surfaces below pH 6.0. This condition would allow penetration of the protein into the hydrophobic core of membrane. With the aid of circular dichroism and Fourier transform infrared spectroscopy, it was determined that the changes in hydrophobicity of AnxVI were accompanied by a decrease of α-helix content, the appearance of new β-sheet structures and an increase of a number of β-turn segments within the AnxVI molecule. Changes of secondary structure of AnxVI were corroborated by an increase in the accessibility of one of the AnxVI tryptophan residues to fluorescence quenchers at pH 4.6, compared with pH 7.4. Our observations provide further insight into some aspects of the molecular interactions between AnxVI and membrane lipid, permitting the suggestion that AnxVI may be involved in some pathological processes where acidification of the cytosol and/or disruption of intracellular ion homeostasis are detected.

show abstract

Section: Discussionmentioning

confidence: 99%

Acidic pH-induced folding of annexin VI is a prerequisite for its insertion into lipid bilayers and formation of ion channels by the protein molecules

et al. 2001

View full text Add to dashboard Cite

show abstract

“…These facts are strong evidences that the enzyme molecules remain in their native state at the interface even upon compression. Denaturation would lead to an alteration on β-sheet structure [33], and therefore would lead to changes in the corresponding bands, which did not occur. The amide I band is highly anisotropic, that is, they are quite sensitive to change in orientation of carbonyl groups, and despite that, only slight modifications are observed in the fingerprint of the bands.…”

Section: Pure Oap At Air-water Interface-irras Spectroscopymentioning

confidence: 97%

Rat osseous plate alkaline phosphatase as Langmuir monolayer—An infrared study at the air–water interface

Caseli

Masui

Furriel

et al. 2008

Journal of Colloid and Interface Science

View full text Add to dashboard Cite

“…Moreover, the enzyme also exhibited at broad high activity (more than 62% maximal activity) at temperatures ranging from 45°to 65°C, which is consistent with the temperature for the survival of Geobacillus species. The purified recombinant Gtd AP was found to retain a relatively high activity after incubation of 60°C for 1 h. This result showed that this enzyme was more stable than AP from shrimp [31], calf intestine [32], Bacillus stearothermophilus [33], and most psychrophiles [8,[34][35][36][37][38]. These thermal sensitive APs significantly lost activity after the incubation at 60°C.…”

Section: Activity Of the G Thermodenitrificans T-2 Apmentioning

confidence: 94%

A Moderately Thermostable Alkaline Phosphatase from Geobacillus thermodenitrificans T2: Cloning, Expression and Biochemical Characterization

Zhang

et al. 2008

Appl Biochem Biotechnol

View full text Add to dashboard Cite

A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (<35% identity), while it exhibits the conservation of the active site and structure element of Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 degrees C, respectively. The enzyme retained a high activity at 45-60 degrees C, while it could be quickly inactivated by a heat treatment at 80 degrees C for 15 min, exhibiting a half-life of 8 min at 70 degrees C. The K(m) and V (max) for pNPP were determined to be 31.5 muM and 430 muM/min at optimal conditions. A divalent cation is essential, with a combination of Mg2+ and Co2+ or Zn2+ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification.

show abstract

Thermal and pH stabilities of alkaline phosphatase from bovine intestinal mucosa: a FTIR study

Cited by 41 publications

References 36 publications

Acidic pH-induced folding of annexin VI is a prerequisite for its insertion into lipid bilayers and formation of ion channels by the protein molecules

Acidic pH-induced folding of annexin VI is a prerequisite for its insertion into lipid bilayers and formation of ion channels by the protein molecules

Rat osseous plate alkaline phosphatase as Langmuir monolayer—An infrared study at the air–water interface

A Moderately Thermostable Alkaline Phosphatase from Geobacillus thermodenitrificans T2: Cloning, Expression and Biochemical Characterization

Contact Info

Product

Resources

About