Nakano A, Marks D L, Tietz P S, de Groen P C, LaRusso N F
Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, MN 55905, USA.
Hepatology. 1995 Jul;22(1):262-6.
The turnover rate of an individual protein is a function of the rates of synthesis and loss of that protein. For most intracellular proteins, loss occurs through digestion by lysosomal or cytosolic proteases. Although a significant proportion of hepatic lysosomal enzymes is released from the hepatocyte by excretion into bile, the contribution of biliary excretion to the turnover of hepatic lysosomal enzymes has never been measured. Thus, we used in vivo pulse-labeling to determine the half-lives of two hepatic hydrolases, beta-galactosidase (beta-gal) and beta-glucuronidase (beta-glu). Each enzyme was purified by immunoisolation from hepatic lysosomes that were isolated at various times after injection of rats with 3H-labeled leucine. The decay curves for the specific radioactivities of beta-gal and beta-glu were used to calculate the half-lives of the proteins, which were 3.8 and 5.1 days, respectively. To determine the percent of total hepatic contents of each enzyme that was lost per day by biliary excretion, we collected bile from bile fistula rats for 24 hours and then used radioimmunoassays to quantitate the amounts of beta-gal and beta-glu in bile and liver samples of the same rats. We found that approximately 4% of the total hepatic contents of both beta-gal and beta-glu was excreted into bile per day. Finally, we used these data to calculate that 31% and 41% of hepatic losses of beta-gal and beta-glu, respectively, were due to biliary excretion. These results suggest that extracellular release through biliary excretion is a major mechanism contributing to the turnover of lysosomal hydrolases.
单个蛋白质的周转率是该蛋白质合成和损失速率的函数。对于大多数细胞内蛋白质而言，损失是通过溶酶体或胞质蛋白酶的消化而发生的。尽管相当一部分肝溶酶体酶通过排泄到胆汁中从肝细胞释放出来，但胆汁排泄对肝溶酶体酶周转率的贡献从未被测量过。因此，我们使用体内脉冲标记来确定两种肝水解酶β-半乳糖苷酶（β-gal）和β-葡萄糖醛酸酶（β-glu）的半衰期。每种酶通过免疫分离从肝溶酶体中纯化，这些溶酶体是在给大鼠注射3H标记的亮氨酸后不同时间分离得到的。β-gal和β-glu的比放射性衰变曲线用于计算蛋白质的半衰期，分别为3.8天和5.1天。为了确定每种酶的肝总含量每天通过胆汁排泄损失的百分比，我们从胆瘘大鼠收集胆汁24小时，然后使用放射免疫测定法定量相同大鼠胆汁和肝脏样品中β-gal和β-glu的量。我们发现，β-gal和β-glu的肝总含量中每天约有4%排泄到胆汁中。最后，我们利用这些数据计算出，β-gal和β-glu肝损失中分别有31%和41%是由于胆汁排泄。这些结果表明，通过胆汁排泄的细胞外释放是溶酶体水解酶周转的主要机制。
