Iwamoto I, Nadel J A, Varsano S, Forsberg L S
Cardiovascular Research Institute, University of California, San Francisco 94143-0130.
Biochim Biophys Acta. 1988 Sep 8;966(3):336-46. doi: 10.1016/0304-4165(88)90083-9.
We studied the metabolism of sulfated cell-surface macromolecules in dog tracheal epithelial cells in primary culture. To examine the time-course and rate of appearance of sulfated macromolecules at the cell surface, the cells were pulsed with 35SO4 for short periods (5-15 min), and the incubation medium was sampled for spontaneously released macromolecules (basal secretions) and for release induced by trypsin (trypsin-accessible secretions). Trypsin-accessible 35S-labeled macromolecules appeared on the cell surface within 5-10 min, increased linearly, and plateaued by 40 min; the median transit time for 35S-labeled macromolecules to reach the cell surface was 21 min. 35S-labeled macromolecules in basal secretions increased with a similar time-course, reaching a plateau by 40 min. Incorporation of [3H]serine into the protein moiety of trypsin-accessible macromolecules occurred more slowly; trypsin-accessible 3H-labeled macromolecules were barely detectable at 1 h and increased to a maximum after 2 h, suggesting the presence of a preformed pool of nonsulfated core protein. Pretreatment with cycloheximide, an inhibitor of protein synthesis, decreased trypsin-accessible 35S-labeled macromolecules log-linearly depending on the duration of pretreatment providing an estimate of the rate of depletion of the core protein pool (t1/2 = 32 min). During continuous exposure to 35SO4, 35S-labeled macromolecules accumulated on the cell surface (trypsin-accessible compartment) for 16 h, at which point the cell-surface pool was saturated (t1/2 = 7.5 h). After pulse-labeling the cells with 35SO4 for 15 min, the 35S-labeled macromolecules disappeared continuously from the cell surface (t1/2 = 4.6 h), and 79% of the radioactivity was recovered in the medium as nondialyzable macromolecules. Release of the 35S-labeled macromolecules from the cell surface was abolished at 4 degrees C, indicative of an energy-dependent process, but multiple proteinase inhibitors did not affect the release. We conclude that sulfate is metabolized rapidly into epithelial cell-surface macromolecules, which accumulate continuously into a relatively large cell-surface pool, before they are released by an undefined energy-dependent mechanism.
我们研究了原代培养的犬气管上皮细胞中硫酸化细胞表面大分子的代谢情况。为了检测硫酸化大分子在细胞表面出现的时间进程和速率，细胞用35SO4短时间脉冲处理（5 - 15分钟），然后采集培养液样本，分析自发释放的大分子（基础分泌）以及胰蛋白酶诱导释放的大分子（胰蛋白酶可及性分泌）。胰蛋白酶可及的35S标记大分子在5 - 10分钟内出现在细胞表面，呈线性增加，40分钟时达到平台期；35S标记大分子到达细胞表面的中位转运时间为21分钟。基础分泌中的35S标记大分子以类似的时间进程增加，40分钟时达到平台期。[3H]丝氨酸掺入胰蛋白酶可及大分子的蛋白质部分的过程较为缓慢；1小时时几乎检测不到胰蛋白酶可及的3H标记大分子，2小时后增加到最大值，这表明存在一个预先形成的非硫酸化核心蛋白池。用蛋白质合成抑制剂环己酰亚胺预处理，根据预处理时间长短，胰蛋白酶可及的35S标记大分子呈对数线性减少，由此可估算核心蛋白池的消耗速率（t1/2 = 32分钟）。在持续暴露于35SO4的过程中，35S标记大分子在细胞表面（胰蛋白酶可及部分）积累16小时，此时细胞表面池饱和（t1/2 = 7.5小时）。用35SO4对细胞进行15分钟脉冲标记后，35S标记大分子持续从细胞表面消失（t1/2 = 4.6小时），79%的放射性以非透析性大分子的形式在培养液中回收。35S标记大分子从细胞表面的释放在4℃时被阻断，表明这是一个能量依赖过程，但多种蛋白酶抑制剂并不影响释放。我们得出结论，硫酸盐迅速代谢为上皮细胞表面大分子，这些大分子持续积累形成一个相对较大的细胞表面池，然后通过一种未知的能量依赖机制释放。
