气道平滑肌中肌醇1,4,5-三磷酸立体特异性结合位点的表征

Characterisation of stereospecific binding sites for inositol 1,4,5-trisphosphate in airway smooth muscle.

Chilvers E R, Challiss R A, Willcocks A L, Potter B V, Barnes P J, Nahorski S R

Department of Pharmacology and Therapeutics, University of Leicester.

Br J Pharmacol. 1990 Feb;99(2):297-302. doi: 10.1111/j.1476-5381.1990.tb14698.x.

Abstract

A 'P2' membrane fraction of bovine tracheal smooth muscle displays high affinity (KD 3.8 +/- 0.2 nM), saturable (Bmax 1003 +/- 170 fmol mg-1 protein) and reversible binding of D-myo[3H]-inositol 1,4,5-trisphosphate ([3H]-Ins(1,4,5)P3). 2. This binding site shows strict stereo- and positional specificity for the D-Ins(1,4,5)P3 isomer with L-Ins(1,4,5)P3, DL-Ins(1,3,4,5)P4 and D-Ins(1,3,4)P3 displacing [3H]-Ins(1,4,5)P3 with Ki values of 20 microM, 0.35 microM and 2.4 microM, respectively. 3. Specific binding of [3H]-Ins(1,4,5)P3 is enhanced at alkaline pH values (maximal at pH 7.75) and, in distinct contrast to [3H]-Ins(1,4,5)P3 binding in rat cerebellum membranes, is not inhibited by Ca2+ (5-500 microM). 4. Heparin displaces [3H]-Ins(1,4,5)P3 specific binding with an IC50 of 7.6 +/- 1.0 micrograms ml-1. 5. Comparative studies demonstrated specific [3H]-Ins(1,4,5)P3 binding in bovine cardiac atrial preparations (Bmax 75 +/- 5 fmol mg-1 protein) and very low specific [3H]-Ins(1,4,5)P3 binding in bovine cardiac ventricle and skeletal muscle membranes (less than or equal to 25 fmol mg-1 protein).

摘要

牛气管平滑肌的“P2”膜组分对D-肌醇[3H]-1,4,5-三磷酸（[3H]-Ins(1,4,5)P3）表现出高亲和力（KD 3.8±0.2 nM）、可饱和性（Bmax 1003±170 fmol mg-1蛋白质）和可逆结合。2. 该结合位点对D-Ins(1,4,5)P3异构体具有严格的立体和位置特异性，L-Ins(1,4,5)P3、DL-Ins(1,3,4,5)P4和D-Ins(1,3,4)P3分别以20 microM、0.35 microM和2.4 microM的Ki值取代[3H]-Ins(1,4,5)P3。3. [3H]-Ins(1,4,5)P3的特异性结合在碱性pH值下增强（在pH 7.75时最大），并且与大鼠小脑膜中[3H]-Ins(1,4,5)P3的结合明显不同，不受Ca2+（5 - 500 microM）抑制。4. 肝素以7.6±1.0微克毫升-1的IC50取代[3H]-Ins(1,4,5)P3的特异性结合。5. 比较研究表明，[3H]-Ins(1,4,5)P3在牛心房制剂中有特异性结合（Bmax 75±5 fmol mg-1蛋白质），而在牛心室和骨骼肌膜中的特异性[3H]-Ins(1,4,5)P3结合非常低（小于或等于25 fmol mg-1蛋白质）。