Mechanisms of protein degradation in growing and non-growing L-cell cultures.
Mechanisms of protein degradation in growing and non-growing L-cell cultures.
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生长和非生长 L 细胞培养物中蛋白质降解的机制。
DOI：
10.1042/bj1820847
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
M. J. Sargus
中科院分区：
文献类型：
--
作者：
J. Amenta;M. J. Sargus
用[~(14)C]亮氨酸和[~3H]胸腺嘧啶核苷标记的L细胞分别置于新鲜生长培养液(含10%血清的最低必需培养液)或降解液(最低必需培养液)中培养3d。~(14)C/~H比值在生长培养中保持不变，在固定相培养中下降，表明生长培养中没有蛋白质周转，固定相培养中蛋白质的降解率为0.6%/h。然而，媒体分析表明，14C标记的蛋白质大约正在被降解。生长菌种为1.2%/h，静止相菌种为1.7%/h。进一步的研究表明，在单层中有一个L细胞亚群，大约包括。在原加工程序中损失了20%-30%。单层原位固定回收率接近100%的实验表明，在生长的培养物中观察到的所有蛋白质分解都是由蛋白质降解亚群造成的。NH4Cl只部分抑制了这些培养物中的蛋白质降解，表明在激活的溶酶体-自噬系统中只有一小部分蛋白质降解。NaF对蛋白降解有更有效的抑制作用，但我们不能区分这种作用是对需要ATP的基础周转机制的影响，还是对细胞透明质中未受调节的蛋白酶活性的直接影响。然而，NH4Cl抑制了将细胞置于降解液中时诱导的蛋白分解，表明诱导的蛋白分解是通过自噬系统发生的。我们的结论是，L细胞在蛋白质降解方面至少存在两种状态：(A)积极复制且不降解细胞蛋白质的亚群；(B)由前一亚群衍生的第二亚群，其降解其标记的大部分蛋白质，不能进一步复制，且不在等渗EDTA缓冲溶液中沉淀。此外，增殖的L细胞，当被置于降压液中时，开始通过涉及自噬溶酶体的机制来降解细胞蛋白。
L-cells prelabelled with [14C]leucine and [3H]thymidine were placed in either fresh growth medium (minimal essential medium with 10% serum) or stepdown medium (minimal essential medium) for 3 days. The 14C/3H ratio remained constant in the growing cultures and decreased in the stationary-phase cultures, indicating no protein turnover in growing cultures and a degradative rate of 0.6%/h in the stationary-phase cultures. Media analysis, however, indicated that 14C-labelled proteins were being degraded at approx. 1.2%/h in growing cultures and 1.7%/h in stationary-phase cultures. Additional studies indicated that a subpopulation of L-cells in the monolayer, comprising approx. 20--30% of the total, were lost in the original processing procedure. Experiments in which recoveries approached 100% by fixation of the monolayer in situ indicated that a protein-degrading subpopulation accounted for all the observed proteolysis in the growing cultures. Proteolysis in these cultures was only partially inhibited with NH4Cl, indicating that only a small part of the protein degradation was occurring in an activated lysosomal-autophagic system. NaF produced a more effective inhibition of proteolysis, but we were not able to distinguish whether this effect was on an ATP-requiring basal-turnover mechanism or a direct effect on unregulated activity of proteinases in the cell hyaloplasm. However, NH4Cl inhibited the proteolysis induced when cells were placed in stepdown medium, suggesting that the induced proteolysis was occurring via the autophagic system. We conclude that L-cells exist in at least two states with respect to protein degradation: (a) a subpopulation that is actively replicating and does not degrade cellular proteins, and (b) a second subpopulation of cells, derived from the preceding one, which degraded most of their labelled proteins, are not capable of further replication, and are not sedimented in an iso-osmotic EDTA buffer solution. In addition, proliferating L-cells, when placed in stepdown medium, begin to degrade cell protein through a mechanism involving autophagolysosomes.