力竭游泳诱导的大鼠睾丸金属结合蛋白的初步分离与鉴定

目的初步分离和鉴定力竭运动诱导的大鼠睾丸金属结合蛋白(testis metal-binding proteins,TMB-Ps)。方法 8只雄性SD大鼠一次性力竭游泳运动(8只对照)后6 h取睾丸和肝脏组织,用镉饱和法测定TMBPs和肝脏金属硫蛋白(metallothionein,MT)含量,用tricine-SDS-PAGE方法分离镉饱和法提取液的TMBPs组分和肝脏MT,用液相色谱-串联质谱法(LC-MS-MS)鉴定TMBPs分离条带。结果力竭运动组大鼠TMBPs水平(113.71±11.72)nmol Cd/g testis显著高于安静对照组(87.14±12.72)nmol Cd/g testis(P0.01),肝脏MT表达量(64.70±14.89)μg/g也显著高于安静对照组(7.32±3.31)μg/g(P0.001)。LC-MS-MS对tricine-SDS-PAGE分离的蛋白条带的鉴定结果为:TMBPs含有泛素、铜-锌-超氧化物歧化酶、酪蛋白样磷蛋白等蛋白质,另外还应包括MT。结论TMBPs由一组具有金属结合性、耐热性和诱导性的蛋白质所组成,主要有泛素、超氧化物歧化酶和酪蛋白样磷蛋白。镉饱和法并非测定金属硫蛋白的特异性方法。

Separation and identification of testicular metal-binding proteins induced by exhaustive swimming in rats

Objective To separate and identify the characteristics of testicular metal-binding proteins(TMBPs) induced by exhaustive swimming in rats.Methods Eight male SD rats were subjected to a bout of exhaustive swimming(8 for control) and the testes and liver were taken out 6 hours after the exercise.TMBPs and hepatic metallothionein(MT) were determined by cadmium-saturation assay.The extracts from the testes and liver by cadmium-saturation method were separated by tricine-SDS-PAGE and TMBPs protein bands were identified by LC-MS-MS.Results The results of cadmium-saturation assay showed that the TMBPs content was significantly increased in the exhaustive swimming rats [(113.71±11.72)nmol Cd/g testis] than that in the controls [(87.14±12.72)nmol Cd/g testis](P<0.01),and the hepatic MT was(64.70±14.89)μg/g,significantly higher than the(7.32±3.31)μg/g in the controls(P<0.001).Ubiquitin,superoxide dismutase (Cu-Zn-SOD),casein-like phosphoprotein(CLPH) were identified in TMBPs,and MT was also included.Conclusions TMBPs are mainly composed of ubiquitin,SOD and CLPH,with characteristics of metal binding,heat resistance and induction.The cadmium-saturation assay is not a specific method for MT determination.