Biphasic denaturation of human placental alkaline phosphatase in guanidinium chloride

Hung, Hui‐Chih; Chang, Gu‐Gang

doi:10.1002/(sici)1097-0134(19981001)33:1<49::aid-prot5>3.0.co;2-g

Cited by 11 publications

(6 citation statements)

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“…Denaturation appeared to be monophasic in this study for the two enzymes. This is not in contradiction with the report of Hung and Chang [39] who evidenced a biphasic denaturation of the enzyme because in the first denaturation phase, the enzyme remains fully active, thus this step was not analysed in the present study. The relative resistance of PLAP to denaturation has been known for a long time.…”

Section: Discussioncontrasting

confidence: 64%

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Denier

Biasini

et al. 2002

BMC Biochem

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Background: In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors.

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Section: Discussioncontrasting

confidence: 64%

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Denier

Biasini

et al. 2002

BMC Biochem

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“…We have demonstrated a GdnHCl-induced biphasic unfolding process of the enzyme and also ruled out a salt effect on that biphasic phenomenon (Fig. 2 A, inset graph) (Hung and Chang, 1998). The urea-induced multiphasic unfolding phenomenon of placental alkaline phosphatase changed to monophasic by adding NaCl (83 mM) into the enzyme solutions during urea denaturation (Fig.…”

Section: Intermediate Form During the Urea-induced Unfolding Of Human Placental Alkaline Phosphatase Can Be Stabilized By Guanidinium Chlmentioning

confidence: 53%

“…the fluorescence wavelength shift induced by urea plus GdnHCl was similar to that of the GdnHCl alone (Hung and Chang, 1998). Because 83 mM GdnHCl itself did not induce any unfolding of the enzyme (Hung and Chang, 1998), these results indicated that GdnHCl, but not NaCl, could help to entrap an unfolding intermediate and stabilize it during the unfolding process induced by urea. In the presence of GdnHCl, the [urea] 0.5 were estimated to be 3.9 and 5.2 M for the N 7 I i and I i 7 I iϩ1 processes, respectively.…”

Section: Intermediate Form During the Urea-induced Unfolding Of Human Placental Alkaline Phosphatase Can Be Stabilized By Guanidinium Chlmentioning

confidence: 62%

“…Alkaline phosphatase from Escherichia coli is quite unique in that the enzyme undergoes continued slow structural annealing long after the return of enzyme activity (Subramaniam et al, 1995). We have characterized the chemical stability of the placental alkaline phosphatase in GdnHCl in which a stable unfolding intermediate was identified (Hung and Chang, 1998). To further characterize the unfolding intermediate of the placental enzyme, we examined the urea-induced unfolding process.…”

Section: Introductionmentioning

confidence: 99%

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Multiple Unfolding Intermediates of Human Placental Alkaline Phosphatase in Equilibrium Urea Denaturation

Hung

Chang

2001

Biophysical Journal

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Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally stable toward a chemical denaturant.

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“…The conformational stability of human placental alkaline phosphatase around the active site region is studied by monitoring the enzyme activity change in the GdmCl‐induced denaturation (Hung and Chang 1998). In the absence of phosphate and magnesium ion, the M3‐free enzyme was found to be sensitive to denaturant (Fig.…”

Section: Resultsmentioning

confidence: 99%

Differentiation of the slow‐binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase

Hung

Chang

2001

Protein Science

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The binding mechanism of Mg 2+ at the M3 site of human placental alkaline phosphatase was found to be a slow-binding process with a low binding affinity (K Mg(app.) ‫ס‬ 3.32 mM). Quenching of the intrinsic fluorescence of the Mg 2+ -free and Mg 2+ -containing enzymes by acrylamide showed almost identical dynamic quenching constant (K sv ‫ס‬ 4.44 ± 0.09 M −1 ), indicating that there is no gross conformational difference between the M3-free and the M3-Mg 2+ enzymes. However, Zn 2+ was found to have a high affinity with the M3 site (K Zn(app.) ‫ס‬ 0.11 mM) and was observed as a time-dependent inhibitor of the enzyme. The dependence of the observed transition rate from higher activity to lower activity (k obs ) at different zinc concentrations resulted in a hyperbolic curve suggesting that zinc ion induces a slow conformational change of the enzyme, which locks the enzyme in a conformation (M3Ј-Zn) having an extremely high affinity for the Zn 2+ (K* Zn(app.) ‫ס‬ 0.33 M). The conformation of the M3Ј-Zn enzyme, however, is unfavorable for the catalysis by the enzyme. Both Mg 2+ activation and Zn 2+ inhibition of the enzyme are reversible processes. Structural information indicates that the M3 site, which is octahedrally coordinated to Mg 2+ , has been converted to a distorted tetrahedral coordination when zinc ion substitutes for magnesium ion at the M3 site. This conformation of the enzyme has a small dynamic quenching constant for acrylamide (K sv ‫ס‬ 3.86 ± 0.04 M −1 ), suggesting a conformational change. Both Mg 2+ and phosphate prevent the enzyme from reaching this inactive structure. GTP plays an important role in reactivating the Zn-inhibited enzyme activity. We propose that, under physiological conditions, magnesium ion may play an important modulatory role in the cell for protecting the enzyme by retaining a favorable geometry of the active site needed for catalysis.Keywords: Slow binding; magnesium ion; zinc ion; enzyme regulation; alkaline phosphatase Alkaline phosphatase (EC 3.1.3.1) is a substrate nonspecific phosphomonoesterase, which exists in almost all living forms, from bacteria to human (McComb et al. 1979). It is an enzyme well known for its clinical and diagnostic applications (McComb et al. 1979). However, the physiological role of the enzyme has not been fully elucidated. The crystal structure of the Escherichia coli alkaline phosphatase was first solved by Sowadski et al. (1985) and has been refined Reprint requests to: Dr. Hui-Chih Hung, Department of Biochemistry, National Defense Medical Center, P.O. Box 90048-501 (Neihu), Taipei 114, Taiwan, Republic of China; e-mail: ibio33@ndmctsgh.edu.tw; fax: 886-2-2933-9996.Abbreviations: GdmCl, guanidinium chloride; M3-Mg enzyme (M1,M2-Zn/M3-Mg), native human placental alkaline phosphatase with the M1 and M2 metal sites occupied by Zn 2+ and M3 site by Mg 2+ ; M3-free enzyme (M1,M2-Zn/M3-free), the enzyme with the M1 and M2 metal sites occupied by Zn 2+ but M3 site is free; M3Ј-Zn enzyme (M1,M2,M3Ј-Zn), inactive enzyme with all metal sites oc...

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Biphasic denaturation of human placental alkaline phosphatase in guanidinium chloride

Cited by 11 publications

References 49 publications

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Multiple Unfolding Intermediates of Human Placental Alkaline Phosphatase in Equilibrium Urea Denaturation

Differentiation of the slow‐binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase

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