微量定磷法测定红细胞钙调素含量

Determination of Red Blood Cell Calmodulin Content by Improved Inorganic Phosphate Microanalzsis

磷酸二酯酶在钙调素(CaM)激活下水解cAMP为AMP。后者经蛇毒水解为无机磷酸和腺苷。用孔雀绿显色测定无机磷酸量与标准CaM比较即可测出CaM活性。该法测定CaM灵敏度3.4ng,可测范围3.4—48ng,批间变异系数3.8％—6.4％。 纯化的红细胞(RBC)经超声破碎,沸水浴加热,离心上清与标准CaM所作的磷酸二脂酶平行激活曲线呈正相关(r=0.9900,p<0.0005)。RBC CaM含量测定,批间变异系数11.5％,回收率97.8％±2.0％。本法测得30例正常人RBC CaM含量为6.31±1.5fg/cell,性别之间无显著差异(p>0.5)

CaM stimulats CaM-dependent PDE which converts cAMP to AMP. AMP can be further hydrolyzed into inorganic phosphate and adenosine by snake venum. The CaM can be determined by measuring the amount of inorganic phosphate with malachite green dye method.The sensitivity was 3.4 ng CaM and the workable range was 3.4-48 ng CaM.The coefficient of variation of CaM activation was 3.8%-6.4%. The RBC was isolated from anticoagulant blood by lymphocyte isolating solution and diluted with 6 volumes of borate buffer,sonicated,heated in boiling water bath for 4.5 min and then rapidly cooled in ice-water bath.The CaM in the supernatant was measured.The correlation coefficient of relation curve for purified CaM and CaM extracted from RBC was 0.9900, p<0.0005.The coefficient of variation of CaM from RBC was 11.5%. Recovery was 97.8±2.0% (n = 4) .The RBC CaM content in 30 health subjects was 6.31±1.50 fg/cell.