ACTIN NH2-TERMINAL PROCESSING AND ITS SIGNIFICANCE

肌动蛋白，一种主要的收缩和结构蛋白，在肌肉和 非肌肉细胞，在其NH2末端被加工，在一系列新的 不用于其他蛋白质的反应：乙酰蛋氨酸或 从完整的多肽链中除去乙酰半胱氨酸残基 翻译完成后，新的NH2末端氨基酸是 乙酰化后形成成熟形式的肌动蛋白。这件事的意义 无法理解唯一处理事件。在拟议的研究中，我们 将确定参与乙酰化的要求 从新药中脱除氨基末端蛋氨酸和半胱氨酸残基 网织红细胞裂解制剂中合成的II类肌动蛋白。通过 用35S-蛋氨酸脉冲标记小鼠L细胞，我们将确定 肌动蛋白是否必须经过处理才能进入细胞的 细胞骨架结构。通过脉冲标记BC3H1平滑肌细胞 35S-半胱氨酸，我们将确定I类和II类肌动蛋白是如何 在同一细胞中加工并沉积成细胞骨架结构 同一时间。我们将确定只产生I类的生物体 肌动蛋白可以忠实地处理第二类肌动蛋白，反之亦然。我们会 I类肌动蛋白乙酰甲硫氨酸裂解的纯化和鉴定 从大鼠肝脏中提取酶(S)，制成抗体并用这些抗体测定 在哺乳动物细胞中，是否存在单独的酶来处理I类 和第二类肌动蛋白，以及这些酶与类似的 低等真核生物中的酶。最后，已知肌球蛋白与肌动蛋白结合。 或靠近肌动蛋白NH2末端。我们将制造未经处理的肌动蛋白 无细胞翻译系统，并使用这些系统来确定肌动蛋白的效果 氨基末端处理对肌动蛋白与肌球蛋白S1结合能力的影响 碎片。这些研究将有助于我们理解肌动蛋白的作用。 细胞骨架组装中的加工和肌动蛋白的工作能力 使用肌球蛋白产生收缩力，需要两个过程 适当的细胞功能和生物体的生存能力。

Actin, a major contractile and structural protein in both muscle and nonmuscle cells, is processed at its NH2-terminus in a series of novel reactions not used for other proteins: an acetyl-methionine or acetyl-cysteine residue is removed from a completed polypeptide chain following completion of translation, and the new NH2-terminal amino acid is acetylated to make the mature form of actin. The significance of this unique processing event is not understood. In the proposed research, we will determine the requirement for the involvement of acetylation in removing NH2-terminal methionine and cysteine residues from newly synthesized class II actins in reticulocyte lysate preparations. By pulse-labeling mouse L-cells with 35S-methionine, we will determine whether or not the actin must be processed before it can enter the cell's cytoskeletal structures. By pulse-labeling BC3H1 smooth muscle cells with 35S-cysteine, we will determine how class I and class II actins are processed and deposited into cytoskeletal structures in the same cell at the same time. We will establish whether organisms which make only class I actins can faithfully process class II actins and visa versa. We will purify and characterize the class I actin acetylmethionine cleaving enzyme(s) from rat liver, make antibodies to it and use these to determine whether, in mammalian cells, separate enzymes exist for processing class I and class II actins, and how closely these enzymes are related to similar enzymes in lower eukaryotes. Finally, myosin is known to bind to actin at or near the actin NH2-terminus. We will make unprocessed actins in a cell-free translation system and use these to determine the effect of actin amino-terminal processing on the ability of actin to bind to the myosin S1 fragment. These studies will help us to understand the role of actin processing in cytoskeletal assembly and in the ability of actin to work with myosin to generate contractile force, two processes required for proper cell function and organismic viability.