INHIBITION OF THE LYSOSOMAL PATHWAY OF PROTEIN-DEGRADATION IN ISOLATED RAT HEPATOCYTES BY AMMONIA, METHYLAMINE, CHLOROQUINE AND LEUPEPTIN
INHIBITION OF THE LYSOSOMAL PATHWAY OF PROTEIN-DEGRADATION IN ISOLATED RAT HEPATOCYTES BY AMMONIA, METHYLAMINE, CHLOROQUINE AND LEUPEPTIN
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DOI：
10.1111/j.1432-1033.1979.tb12956.x
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发表时间：
1979-01-01
期刊：
影响因子：
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通讯作者：
SOLHEIM, AE
中科院分区：
文献类型：
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作者：
SEGLEN, PO;GRINDE, B;SOLHEIM, AE
分离的大鼠肝细胞中的蛋白质降解被测量为[14 C]缬氨酸从预标记蛋白质中的释放。为了降低背景放射性，通过在37 ℃连续提取来耗尽细胞内[14 C]缬氨酸库。C，实现细胞内池和不含缬氨酸的细胞外培养基之间的平衡。提取后，保留了一个小的非平衡细胞内[14 C]缬氨酸池;该池仅在存在持续蛋白质合成的情况下才能标记，可能代表来自蛋白质降解的缬氨酸和含缬氨酸寡肽。从降解蛋白质释放的[14 C]缬氨酸未显著再用于细胞内蛋白质合成（放线菌酮或高浓度未标记缬氨酸无影响），反映了蛋白质合成速率低和缬氨酸快速转运至细胞外培养基中，这两种情况均为分离肝细胞的特征。从体内预标记24小时的细胞中，[14 C]缬氨酸以5%/h的线性速率释放，可能代表真实的总体蛋白质降解速率。亲溶酶体抑制剂NH 3（10 mM NH 4Cl）抑制70%的降解，推测是通过溶酶体途径。从1小时预标记的细胞中，[14 C]缬氨酸以下降的速率释放，NH3仅抑制降解45%，表明大多数短寿命蛋白质通过非溶酶体途径降解。氯喹和甲胺，积累在溶酶体中，由于其弱碱性和抑制肝细胞蛋白质降解的程度与NH3相同，没有加和性。这些化合物似乎选择性地和完全地阻断蛋白质降解的溶酶体途径。亮抑酶肽结合并抑制某些溶酶体蛋白酶的活性，也抑制蛋白质降解，几乎达到与NH3相同的程度，但小部分效应（< 20%）是NH3效应的叠加效应，表明对非溶酶体蛋白质降解的轻微抑制。4种抑制剂中，只有NH_3的作用在2 h内迅速可逆。亮抑酶肽是唯一的降解抑制剂，也不影响蛋白质的合成。在无蛋白质培养基中，氯喹浓度> 0.2 mmol/l时可引起显著的细胞死亡，即，在最大抑制蛋白质降解所需的浓度范围内。在缺氧条件下孵育肝细胞导致蛋白质降解的抑制，其大于NH3的作用，并且部分加和于NH3的作用，即，大部分通过溶酶体途径的降解和超过1/2的通过非溶酶体途径的降解似乎是能量依赖性的。
Protein degradation in isolated rat hepatocytes was measured as the release of [14C]valine from prelabeled protein. To reduce background radioactivity, the intracellular [14C]valine pool was depleted by serial extraction at 37.degree. C, effecting equilibration between the intracellular pool and the valine-free extracellular medium. After extraction, a small, non-equilibrating intracellular [14C]valine pool remained; this pool could only be labeled in the presence of ongoing protein synthesis and might represent valine and valine-containing oligopeptides derived from protein degradation. The [14C]valine released from degraded protein was not significantly re-utilized for protein synthesis intracellularly (no effect of cycloheximide or high concentrations of unlabeled valine), reflecting the low rate of protein synthesis and the rapid transport of valine into the extracellular medium, both characteristic of isolated hepatocytes. From cells prelabeled for 24 h in vivo, [14C]valine was released at a linear rate of 5%/h, probably representing the true overall protein degradation rate. The lysosomotropic inhibitor NH3 (10 mM NH4Cl) inhibited 70% of the degradation, presumably by the lysosomal pathway. From 1-h pre-labeled cells, [14C]valine was released at a declining rate and NH3 inhibited degradation by only 45%, suggesting that most short-lived proteins are degraded by the non-lysosomal pathway(s). Chloroquine and methylamine, accumulated in lysosomes due to their weak base properties and inhibited hepatocytic protein degradation to the same extent as NH3, with no additivity. These compounds seem to block the lysosomal pathway of protein degradation selectively and completely. Leupeptin, which binds to and inhibits the activity of certain lysosomal proteases, also inhibited protein degradation almost to the same extent as NH3, but with a small part of the effect (< 20%) being additive to the NH3 effect and suggesting a slight inhibition of non-lysosomal protein degradation. Of 4 inhibitors tested, only the effect of NH3 was rapidly reversible within 2 h. Leupeptin was the only degradation inhibitor which did not also affect protein synthesis. Chloroquine caused significant cell death at concentrations > 0.2 mmol/l in protein-free medium, i.e., in the concentration range needed for maximal inhibition of protein degradation. Incubation of hepatocytes under anoxic conditions resulted in inhibition of protein degradation which was greater than, and partially additive to, the effect of NH3, i.e., most of the degradation by the lysosomal pathway and more than 1/2 of the degradation by non-lysosomal pathways appears to be energy-dependent.