Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7-11 as the essential pentapeptide.
Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7-11 as the essential pentapeptide.
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DOI：
10.1016/s0021-9258(19)62694-3
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发表时间：
1986-05
期刊：
影响因子：
--
通讯作者：
J. Dice;H. Chiang;E P Spencer;J M Backer
中科院分区：
文献类型：
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作者：
J. Dice;H. Chiang;E P Spencer;J M Backer
我们已经确定了显微注射核糖核酸酶A的一个五肽区域，这是在血清提取过程中促进该蛋白降解所必需的。我们用红细胞介导的微量注射和渗透裂解法将还原甲基化的核糖核酸酶A、核糖核酸酶S蛋白(残基21-124)和核糖核酸酶S多肽(残基1-20)引入人成纤维细胞胞浆。撤除血清后，核糖核酸酶A和核糖核酸酶S肽的降解率增加2倍，而核糖核酸酶S蛋白的分解代谢不受此调节。核糖核酸酶S肽的某些片段也以血清依赖的方式降解(残基1-14和4-13)，而其他片段不能(残基1-10和2-8)。核糖核酸酶S肽在装载到红细胞幽灵中时被切割成两个较小的放射性多肽。在8M尿素存在下，根据Sephadex柱层析测定的相对分子质量，结合连续Edman降解，我们初步将较大的片段鉴定为7-11残基，以确定放射性赖氨酸的位置。较小的多肽片段似乎是氨基末端二肽Lys-Glu和/或残基7-8，Lys-Phe。显微注射入成纤维细胞后，在无血清的情况下，五肽的降解速度加快，而二肽的降解不受影响。我们通过合成S五肽，并通过还原甲基化标记它，证实了7-11残基是该五肽较大的水解物。在8M尿素中葡聚糖凝胶层析后，它在预期的位置迁移，在装载到红细胞中时仅被轻微地水解。最后，注射入成纤维细胞后，这种五肽的降解在血清撤除时增加了2倍。这些结果，结合我们最近的其他研究(McEligott，M.A.，Miao，P.和Dice，J.F.(1985)J.Biol)。化学。260,11986-11993)，表明五肽，赖氨酸-苯丙氨酸-谷氨酸-精氨酸-谷氨酰胺，靶向微量注射核糖核酸酶A到溶酶体，以促进血清剥夺时的降解。
We have identified a pentapeptide region of microinjected ribonuclease A that is required for enhanced degradation of this protein during serum withdrawal. We introduced reductively methylated [3H]ribonuclease A, [3H]ribonuclease S-protein (residues 21-124), and [3H]ribonuclease S-peptide (residues 1-20) into the cytosol of human fibroblasts by red cell-mediated microinjection and osmotic lysis of pinosomes. The degradative rates of ribonuclease A and ribonuclease S-peptide are increased 2-fold upon withdrawal of serum, while catabolism of ribonuclease S-protein is not regulated in this manner. Certain fragments of ribonuclease S-peptide are also degraded in a serum-dependent fashion (residues 1-14 and 4-13), while other fragments are not (residues 1-10 and 2-8). [3H]Ribonuclease S-peptide is cleaved into two smaller radioactive peptides during loading into red cell ghosts. We tentatively identified the larger fragment as residues 7-11 based on its molecular weight determined by Sephadex chromatography in the presence of 8 M urea combined with sequential Edman degradation to identify the position of radioactive lysines. The smaller peptide fragment appears to be the amino-terminal dipeptide, Lys-Glu, and/or residues 7-8, Lys-Phe. After microinjection into fibroblasts, the pentapeptide is degraded at an enhanced rate in the absence of serum, while degradation of the dipeptide is not affected. We confirmed that residues 7-11 constitute the larger hydrolysis product of S-peptide by synthesizing this pentapeptide and radiolabeling it by reductive methylation. It migrated at the expected position after Sephadex chromatography in 8 M urea and was further hydrolyzed only slightly during loading into red cells. Finally, degradation of this pentapeptide after injection into fibroblasts was enhanced 2-fold upon serum withdrawal. These results, combined with our other recent studies (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993), suggest that the pentapeptide, Lys-Phe-Glu-Arg-Gln, targets microinjected ribonuclease A to lysosomes for enhanced degradation during serum deprivation.