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Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome.

Denier CC, Brisson-Lougarre AA, Biasini GG, Grozdea JJ, Fournier DD - BMC Biochem. (2002)

Bottom Line: 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.This proportion did not significantly increase with normal pregnancy.By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Synthèse et Physicochimie de Molécules d'Intérêt Biologique, Université Paul Sabatier (UMR 5068), 118 route de Narbonne, 31 062, Toulouse, France. denier@cict.fr

ABSTRACT

Background: In humans, there are four alkaline phosphatases, and each form exhibits 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 pregnant 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.

Results: To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity.

Conclusion: Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

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Inhibitors and substrate used.
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Figure 1: Inhibitors and substrate used.

Mentions: Kinetic measurements were performed in triplicate at 37°C, at pH 9.5 in 0.1 mol/L diethanolamine-HCl buffer containing 45 mmol/L MgCl2, with an ionic strength maintained constant (0.2) with NaCl, using sodium p-nitrophenylphosphate as substrate. The amount of reaction product (paranitrophenate) was quantified using absorption at 400 nm (apparent ε = 18500 L.mol-1.cm-1 at pH 9.5). All the kinetics were carried out for at least 5 minutes and the initial velocities determined by the slopes using the software included in the Perkin Elmer UV-Visible spectrophotometer Lamda 15. The reversible inhibitors sodium thiophosphate and L-p-bromotetramisole (2,3,5,6-tetrahydro-6-phenylimidazo-[2,1-b]-thiazol; Fig. 1) were incubated for five minutes with the enzyme before the addition of substrate. Data were analysed by multiple nonlinear regression using a non-linear global optimization method based on simulated annealing (J. Czaplicki, V. Marcel and D. Fournier, manuscript in preparation) with equations solved according to the rapid equilibrium hypothesis.


Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome.

Denier CC, Brisson-Lougarre AA, Biasini GG, Grozdea JJ, Fournier DD - BMC Biochem. (2002)

Inhibitors and substrate used.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC65044&req=5

Figure 1: Inhibitors and substrate used.
Mentions: Kinetic measurements were performed in triplicate at 37°C, at pH 9.5 in 0.1 mol/L diethanolamine-HCl buffer containing 45 mmol/L MgCl2, with an ionic strength maintained constant (0.2) with NaCl, using sodium p-nitrophenylphosphate as substrate. The amount of reaction product (paranitrophenate) was quantified using absorption at 400 nm (apparent ε = 18500 L.mol-1.cm-1 at pH 9.5). All the kinetics were carried out for at least 5 minutes and the initial velocities determined by the slopes using the software included in the Perkin Elmer UV-Visible spectrophotometer Lamda 15. The reversible inhibitors sodium thiophosphate and L-p-bromotetramisole (2,3,5,6-tetrahydro-6-phenylimidazo-[2,1-b]-thiazol; Fig. 1) were incubated for five minutes with the enzyme before the addition of substrate. Data were analysed by multiple nonlinear regression using a non-linear global optimization method based on simulated annealing (J. Czaplicki, V. Marcel and D. Fournier, manuscript in preparation) with equations solved according to the rapid equilibrium hypothesis.

Bottom Line: 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.This proportion did not significantly increase with normal pregnancy.By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Synthèse et Physicochimie de Molécules d'Intérêt Biologique, Université Paul Sabatier (UMR 5068), 118 route de Narbonne, 31 062, Toulouse, France. denier@cict.fr

ABSTRACT

Background: In humans, there are four alkaline phosphatases, and each form exhibits 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 pregnant 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.

Results: To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity.

Conclusion: Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

Show MeSH
Related in: MedlinePlus