Measurement of protein synthesis in rat lungs perfused in situ.
Measurement of protein synthesis in rat lungs perfused in situ.
复制标题
原位灌注大鼠肺中蛋白质合成的测量。
作者：
C. A. Watkins;D. Rannels
研究了氨基酸的区室化，以确定准确测量原位灌注大鼠肺中蛋白质合成速率所需的条件。用含有4.5%（w/v）牛血清白蛋白、5.6mM-葡萄糖、正常血浆浓度的19种氨基酸和8.6- 690 μ m-[U-（14）C]苯丙氨酸的Krebs-Henseleit碳酸氢盐缓冲液灌注肺。灌注液用与肺通气相同的加湿气体混合物[O（2）/CO（2）（19：1）或O（2）/N（2）/CO（2）（4：15：1）]平衡。[U-（14）C]苯丙氨酸被证明是研究灌注肺中蛋白质合成的合适前体：它迅速进入组织（t（（1/2）），81 s），并且不转化为其他化合物。当灌流液苯丙氨酸降低到正常血浆浓度的5倍以下时，作为蛋白质合成前体的苯丙氨酸池的比放射性下降，因此[（14）C]苯丙氨酸掺入蛋白质中的比放射性下降。相反，[（14）C]组氨酸掺入肺蛋白不受影响。在低灌注液苯丙氨酸浓度下，基于苯丙氨酰-tRNA的比放射性的蛋白质合成速率介于从细胞外或细胞内池中苯丙氨酸的比放射性计算的速率之间。当细胞外苯丙氨酸增加时，基于这三个苯丙氨酸池的特定放射性的速率是相同的。这些意见表明：（1）苯丙氨酸在肺组织中是区室化的，（2）细胞外和细胞内总的苯丙氨酸池都不是蛋白质前体的唯一来源，（3）在低细胞外苯丙氨酸浓度下，如果用游离氨基酸的比放射性计算蛋白质合成速率是错误的，（4）在低细胞外苯丙氨酸浓度下，蛋白质合成速率是错误的。（4）细胞外苯丙氨酸浓度高时，区室化的影响可以忽略不计，根据游离或tRNA结合的苯丙氨酸库的比放射性可以准确地计算蛋白质合成。
Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs-Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6-690mum-[U-(14)C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O(2)/CO(2) (19:1) or O(2)/N(2)/CO(2) (4:15:1)]. [U-(14)C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t((1/2)), 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [(14)C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [(14)C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.