铅对人类胎盘碱性磷酸酶活力与构象的影响

THE EFFECT OF LEAD NITRATE ON CONFORMATION AND CATALYTIC ACTIVITY OF HUMAN PLACENTAL ALKALINE PHOSPHATASE

目的研究铅对人类胎盘碱性磷酸酶（ＰＬＡＰ）的作用，以探讨铅影响胎儿发育的分子机制。②方法应用分光光度法测定不同浓度Ｐｂ（ＮＯ３）２存在下的ＰＬＡＰ活力，用荧光法检测其荧光光谱的变化；用双倒数作图法确定Ｐｂ（ＮＯ３）２对该酶的抑制类型。③结果低浓度的Ｐｂ（ＮＯ３）２（＜０．５０ｍｍｏｌ／Ｌ）对酶活力无明显影响，但其内源荧光强度略有增加，发射峰位蓝移；当Ｐｂ（ＮＯ３）２的浓度为０．７５ｍｍｏｌ／Ｌ时，酶活力及其荧光强度均迅速下降；当Ｐｂ（ＮＯ３）２浓度＞０．７５ｍｍｏｌ／Ｌ时，随其浓度的增加，酶活力与其荧光强度逐渐降低，但发射峰位蓝移减少；当Ｐｂ（ＮＯ３）２浓度为１０．００ｍｍｏｌ／Ｌ时，酶活力丧失，荧光熄灭。Ｐｂ（ＮＯ３）２是ＰＬＡＰ的反竞争性抑制剂，其抑制常数为１．４０ｍｍｏｌ／Ｌ．④结论Ｐｂ（ＮＯ３）２抑制了ＰＬＡＰ的活力，并使其构象发生了改变，这可能是铅影响胎儿发育的重要分子机制之一。

Objective To study the effect of lead on human placental alkaline phosphatase(PLAP)and discuss the molecular machanism of lead affecting the development of fetus. Methods Spectrophotometry was used to study the activity of PLAP under different concentrations of Pb(NO_3)_2.Fluorometry was used to observe the change of the fluorescence spectra.The inhibition type of Pb(NO_3)_2 on PLAP was determined by using the double reciprocal plot. Results No obvious change of PLAP activity was observed at the concentrations of Pb(NO_3)_2 lower than 0.5mmol/L,whereas its fluorescence emission intensity was slightly increased and its emission maximum was blueshifted.When the concentration of Pb(NO_3)_2 was 0.75mmol/L,both the PLAP activity and emission intensity were rapidly decreased. When the concentration of Pb(NO_3)_2 was higher than 0.75mmol/L, the PLAP activity and emission maximum were gradually decreased, while the emission maximum was less blueshifted.At concentration of 10.00mmol/L, the PLAP activity was completely lost and the fluorescence was quenched. Pb(NO_3)_2 was a uncompetitive inhibitor of PLAP and its inhibition constant (Ki) is 1.40mmol/L. Conclusion The PLAP activity was inhibited and its conformation was changed by Pb(NO_3)_2.This may be one of the important molecular mechanisms that Pb(NO_3)_2 influences the development of fetus. 