MICROINJECTION STUDIES ON PROTEIN DEGRADATION

蛋白质降解的各个方面将使用红细胞介导的方法进行研究 向培养的哺乳动物细胞中微量注射蛋白质。我们将决定 不同的蛋白质亚基是否以相同的速度翻转，以及 底物类似物与酶的共价结合延缓了它们的降解。我们 将尝试通过联合注射检测蛋白质降解的初始反应 或通过将注射的细胞暴露于低浓度的 温度。为了确定溶酶体是否参与了细胞的降解 我们将不可降解的D-氨基酸肽偶联到蛋白质上 在注射之前。未降解的多肽预计会在 如果蛋白质在那里发生降解，就会产生溶酶体。我们还建议将 突变的HeLa细胞以较低的速率降解免疫球蛋白G。

Various aspects of protein degradation will be studied using red cell-mediated microinjection of proteins into cultured mammalian cells. We will determine whether dissimilar subunits of proteins turn over at identical rates and whether covalent-binding of substrate analogs to enzymes retards their degradation. We will attempt to detect initial reactions in protein degradation by coinjection of protease inhibitors and labeled proteins or by exposing injected cells to low temperature. To determine whether lysosomes are involved in the degradation of soluble proteins we will couple nondegradable D-amino acid peptides to proteins prior to injection. Non-degraded peptides are expected to accumulate within the lysosomes if degradation of protein occurs there. We also propose to isolate mutant HeLa cells that degrade immunoglobin G at reduced rates.