MICROINJECTION STUDIES ON PROTEIN DEGRADATION

蛋白质降解的各个方面将使用红细胞介导的 将蛋白质显微注射到培养的哺乳动物细胞中。 我们将确定 蛋白质的不同亚基是否以相同的速率转变， 底物类似物与酶的共价结合延缓了它们的降解。 我们 将尝试通过共注射检测蛋白质降解的初始反应 蛋白酶抑制剂和标记的蛋白质，或通过将注射的细胞暴露于低浓度 温度 为了确定溶酶体是否参与降解 可溶性蛋白质我们将偶联不可降解的D-氨基酸肽蛋白质 注射前。 预期未降解的肽在细胞内积累， 如果蛋白质降解发生在溶酶体。 我们还建议隔离 以降低的速率降解免疫球蛋白G的突变HeLa细胞。

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.