Role of metal ions on the secondary and quaternary structure of alkaline phosphatase from bovine intestinal mucosa

Bortolato, Muriel; Besson, Françoise; Roux, B.

doi:10.1002/(sici)1097-0134(19991101)37:2<310::aid-prot16>3.0.co;2-b

Cited by 42 publications

(8 citation statements)

References 56 publications

(83 reference statements)

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“…These findings suggest similar association rates constants in all cases, i.e. full active site access, even though the profile for IAP suggests additional conformational changes occurring in demetalated IAP [33]. To minimize protein monomerization and incomplete recovery during IAP remetalation, reconstitution was initiated before full enzyme inactivation was achieved, explaining the residual activity at 0.01 µM Zn 2+ in Figure 4 .…”

Section: Resultsmentioning

confidence: 77%

Catalytic Signature of a Heat-Stable, Chimeric Human Alkaline Phosphatase with Therapeutic Potential

et al. 2014

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Recombinant alkaline phosphatases are becoming promising protein therapeutics to prevent skeletal mineralization defects, inflammatory bowel diseases, and treat acute kidney injury. By substituting the flexible crown domain of human intestinal alkaline phosphatase (IAP) with that of the human placental isozyme (PLAP) we generated a chimeric enzyme (ChimAP) that retains the structural folding of IAP, but displays greatly increased stability, active site Zn2+ binding, increased transphosphorylation, a higher turnover number and narrower substrate specificity, with comparable selectivity for bacterial lipopolysaccharide (LPS), than the parent IAP isozyme. ChimAP shows promise as a protein therapeutic for indications such as inflammatory bowel diseases, gut dysbioses and acute kidney injury.

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Section: Resultsmentioning

confidence: 77%

Catalytic Signature of a Heat-Stable, Chimeric Human Alkaline Phosphatase with Therapeutic Potential

et al. 2014

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“…In general, the protein samples migrated in tighter bands when compared to BN-PAGE and to distances more consistent with their native molecular weights. 33, 36, 37 When each of the proteins subjected to NSDS-PAGE was assayed for enzymatic activity, all displayed significant functionality, comparable to their catalytic activity after BN-PAGE ( Figure 3a-d , right). As expected, these enzymatic functions were not observed after subjecting the enzymes to denaturing SDS-PAGE.…”

Section: Resultsmentioning

confidence: 96%

Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions

2014

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Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used to obtain high resolution separation of complex mixtures of proteins. The method initially denatures the proteins that will undergo electrophoresis. Although covalent structural features of resolved proteins can be determined with SDS-PAGE, functional properties are destroyed, including the presence of non-covalently bound metal ions. To address this shortcoming, blue-native (BN)-PAGE has been introduced. This method retains functional properties but at the cost of protein resolving power. To address the need for a high resolution PAGE method that results in the separation of native proteins, experiments tested the impact of changing the conditions of SDS-PAGE on the quality of protein separation and retention of functional properties. Removal of SDS and EDTA from the sample buffer together with omission of a heating step had no effect on the results of PAGE. Reduction of SDS in the running buffer from 0.1% to 0.0375% together with deletion of EDTA also made little impact on the quality of the electrophoretograms of fractions of pig kidney (LLC-PK1) cell proteome in comparison with that achieved with the SDS-PAGE method. The modified conditions were called native (N)SDS-PAGE. Retention of Zn2+ bound in proteomic samples increased from 26 to 98% upon shifting from standard to modified conditions. Moreover, seven of nine model enzymes, including four Zn2+ proteins that were subjected to NSDS-PAGE retained activity. All nine were active in BN-PAGE, whereas all underwent denaturation during SDS-PAGE. Metal retention after electrophoresis was additionally confirmed using laser ablation-inductively coupled plasma-mass spectrometry and in-gel Zn-protein staining using the fluorophore TSQ.

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“…Dut N FKW displayed a gain of α-helical content, perhaps due to stabilization of the α2–α3 loop, whereas the other mutants displayed a net loss of α-helical content (Table 1; Fig. 2A), perhaps due to loss of Mg 2+ [38, 39]. However, CD spectrum analysis is highly reliant on protein concentration, and since some of the mutants were prone to aggregation (most notably Dut N MgB and Dut N MgAB , which displayed the greatest loss of α-helical structure), the determined concentrations may not have been accurate.…”

Section: Resultsmentioning

confidence: 99%

Derepression of SaPIbov1 Is Independent of φNM1 Type 2 dUTPase Activity and Is Inhibited by dUTP and dUMP

Hill

Vlach

Parker

et al. 2017

Journal of Molecular Biology

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Staphylococcus aureus is an opportunistic human pathogen able to transfer virulence genes to other cells through the mobilization of S. aureus pathogenicity islands (SaPIs). SaPIs are derepressed and packaged into phage-like transducing particles by helper phages like 80α or φNM1. Phages 80α and φNM1 encode structurally distinct dUTPases, Dut80α (type 1) and DutNM1 (type 2). Both dUTPases can interact with the SaPIbov1 Stl master repressor, leading to derepression and mobilization. That two structurally distinct dUTPases bind the same repressor led us to speculate that dUTPase activity may be important to the derepression process. In type 1 dUTPases, Stl binding is inhibited by dUTP. The purpose of this study was to assess the involvement of dUTP binding and dUTPase activity in derepression by DutNM1. DutNM1 activity mutants were created and tested for dUTPase activity using a novel NMR-based assay. We found that all DutNM1 null activity mutants interacted with the SaPIbov1 Stl C-terminal domain, formed DutNM1–Stl heterodimers, and caused release of the Pstr promoter. However, promoter release was inhibited in the presence of dUTP or dUMP. We tested two φNM1 mutant phages that had null enzyme activity and found they could still mobilize SaPIbov1. These results show that only the apo form of DutNM1 is active in Stl derepression, and that dUTPase activity is not necessary for mobilization of SaPIbov1 by DutNM1.

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Role of metal ions on the secondary and quaternary structure of alkaline phosphatase from bovine intestinal mucosa

Cited by 42 publications

References 56 publications

Catalytic Signature of a Heat-Stable, Chimeric Human Alkaline Phosphatase with Therapeutic Potential

Catalytic Signature of a Heat-Stable, Chimeric Human Alkaline Phosphatase with Therapeutic Potential

Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions

Derepression of SaPIbov1 Is Independent of φNM1 Type 2 dUTPase Activity and Is Inhibited by dUTP and dUMP

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