摘要
利用ＤＰＡ使Ｔｂ３＋的荧先强度显著增强的原理进行带３蛋白活性的测定，方法简便、灵敏、重复性好，而且能够进行连续荧光扫描测量．应用连续荧光扫描法测定了带３蛋白介导的ＤＰＡ与Ｃｌ－交换的动力学特征参数．结果表明，带３蛋白介导的ＤＰＡ与Ｃｌ－的交换对ＤＩＤＳ非常敏感，受ＤＩＤＳ的强烈抑制，抑制程度大于９０％；ＤＰＡ由内向外转运的米氏常Ｋｍ＝２８．１—３１．２ｍｍｏｌ／Ｌ；带３蛋白的天然底物Ｃｌ－从内侧竞争性抑制ＤＰＡ向外转运，抑制常数ｋｉ＝６０．４±６．９ｍｍｏｌ／Ｌ；膜内侧ＤＰＡ与外侧Ｃｌ－交换的活化能，在４—２５℃范围内为５．８±０．５ｋＣａｌ／ｍｏｌ２５—３７℃范围内为１９．８±１．５ｋＣａｌ／ｍｏｌ；膜内侧ＤＰＡ与外侧Ｃｌ－交换受转运介质ｐＨ（膜内外对称改变）的显著影响，ｐＨ＜７．４时，交换速度显著升高．本实验证明ＤＰＡ确是经带３蛋白而转运的，但转运机制可能与无机离子转运有所不同。
The greatly enhanced fluorescence of Tb3+, when complexed with dipicolinic acid DPA),affords a simple and highly sensitive method for monitoring continuous anion flux through the erythrocyte anion exchanger, band 3 protein. With this method,the kinetics of DPA-Cl- heteroexchange in resealed red cell ghost was studied in detail. The results are as following. (1) The heteroexchange of DPA-Cl- is almost blocked completely by DIDS (>90%), a typical band 3 inhibitor; (2) The Michaelis constant for the inner tiansport site of band 3 protein is in the range of 28.1 to 31.2mmol/L, (3) The natural substrate of band 3Cl-, inhibited the efflux of DPA competitively from inner membrane surface ,displaying the Ki value of 60.4±6.9mmol/L; (4) The activation energy of DPA transport by band 3 is 19.8±1.5kCal/mol in the mperature range of 25 to 37℃ and 5.8±0.5kCal/mol in the range of 4to 25℃, (5) The rate constant of heteroexchange of DPA-Cl- is affected significantly by pH value (the pH values are varied symmetrically inside and outside), and increased markedly when pH value was lower than 7.4. According to these results,it should be safe to conclude that DPA is truly transported via band 3, but the mechanism of DPA tiansport is likely differ ent from that of CL- self-exchange.
出处
《生物物理学报》
CAS
CSCD
北大核心
1994年第2期243-250,共8页
Acta Biophysica Sinica
基金
国家自然科学基金
生物大分子国家开放实验室资助
关键词
带3蛋白
红细胞
血影
动力学
吡啶二羧酸
离子
Band 3 protein Anion transport Red cell ghost Transport kinetics Fluorescent method
