Mortimore G E, Ward W F
J Biol Chem. 1981 Jul 25;256(14):7659-65.
The hypothesis that cytoplasmic protein in rat liver is internalized and degraded intralysosomally during basal or steady state turnover as well as in deprivation-accelerated states was tested by (a) determining the subcellular location of degradable protein in homogenates and (b) comparing the amounts of this protein pool with rates of long lived protein degradation in livers perfused under the above conditions. Greater than 95% of total proteolysis in homogenates, measured at 37 degrees C from free amino acid generation or the release of [14C]valine in previously labeled livers, was associated with particulate fractions, and it paralleled lysosomal markers closely during graded differential centrifugation after Triton WR-1339 loading. Because 14C-labeled cytosolic proteins were not degraded by intact isolated lysosomes at pH 6 or 7, the protein substrate must have been internalized by lysosomes prior to homogenization. Time courses of intralysosomal proteolysis from maximal deprivation down to and including basal were identical in shape, and each leveled off abruptly after 120-150 min. Although the velocities of proteolysis were reduced in vitro, estimates of the degradable protein pools were closely proportional to corresponding rates of long lived degradation in perfused livers. In absolute terms, they fit recent predictions of internalized cytoplasmic protein based on stereological measurements of lysosomal volumes.
(a) 确定匀浆中可降解蛋白的亚细胞定位，以及 (b) 将该蛋白池的量与在上述条件下灌注的肝脏中长寿命蛋白降解速率进行比较。在37摄氏度下，通过游离氨基酸生成或先前标记肝脏中[14C]缬氨酸的释放来测量，匀浆中超过95%的总蛋白水解与颗粒部分相关，并且在Triton WR - 1339加载后的分级差速离心过程中，它与溶酶体标记物密切平行。由于pH 6或7时完整分离的溶酶体不会降解14C标记的细胞溶质蛋白，因此蛋白底物在匀浆之前必定已被溶酶体内化。从最大剥夺状态直至基础状态（包括基础状态）的溶酶体内蛋白水解时间进程形状相同，并且在120 - 150分钟后均突然趋于平稳。尽管体外蛋白水解速度降低，但可降解蛋白池的估计值与灌注肝脏中相应的长寿命降解速率密切成比例。就绝对值而言，它们符合基于溶酶体体积的体视学测量对内化细胞质蛋白的最新预测。
