NEURAL POLYHORMONE PROCESSING MECHANISMS

越来越多的神经分泌肽被发现衍生出来 来自一种多激素前体，其序列中包含 一种以上分泌产物的结构。这提出了根本问题 关于控制生产的生化机制的几个问题 这些多肽及其路由到适当的亚细胞位置。 具体而言，这些函数在多大程度上依赖于 前体，而不是加工酶的性质？这 这个问题将在处理顺序的实验中解决 导致海兔袋中神经分泌型产卵激素(ELH)的原因 细胞。该序列中的前体具有已知的一级结构和 通过蛋白分解过程使ELH和其他分泌肽上升 序列，对其进行了详细表征。此外，中间体 在该序列中，导致不同产品被路由到不同的 单元格内的位置。有许多假定的切割位点 在前体中，但这些化合物中的哪一个实际使用尚不清楚。 此外，前体及其中间体可能是膜结合的， 但这些分子中调节这种联系的区域，可能是 在路由中的重要性，还有待确定。此外，它也不是 但已知前体和中间体是否被相同或相同的 不同的内切酶。这项提案中的实验旨在 回答这些问题。前体的部分序列分析， 中间体和产物将被用来通过 参考先驱体的已知结构。多肽的比较 在无细胞翻译系统中合成的前体的映射到 来自完整细胞合成的前体的那些将识别 包含“信号”序列的区域。脂溶性标记物 光活化探头将获得膜区域的信息 协会。前体解理和中间体解理的表征 袋状细胞匀浆中的活性应揭示多样性的程度 加工内切酶，以及亚细胞分级技术 将被用来确定它们的亚细胞分布。这些 实验应该对基本的细胞过程有深入的了解 控制神经肽和多肽激素的产生。

A growing number of neurosecretory peptides are being found to be derived from a polyhormonal precursor which contains within its sequence the structures of more than one secretory product. This raises fundamental questions concerning the biochemical mechanisms controlling the production of these peptides and their routing to appropriate subcellular locations. Specifically, to what extent do these functions depend on the structure of the precursor, as opposed to properties of the processing enzymes? This question will be addressed in experiments on the processing sequence leading to the neurosecretory egg-laying hormone (ELH) in Aplysia bag cells. The precursor in this sequence is of known primary structure and gives rises to ELH and other secretory peptides by a proteolytic processing sequence which has been characterized in detail. Moreover, intermediates in this sequence which lead to different products are routed to different locations within the cell. There are a number of putative cleavage sites in the precursor, but which of these are actually used is not yet known. Moreover, the precursor and its intermediates are probably membrane-bound, but the regions of these molecules mediating this association, which may be important in routing, have yet to be determined. In addition, it is not yet known if the precursor and intermediates are cleaved by the same or different endoproteases. The experiments in this proposal are designed to answer these questions. Partial sequence analysis of the precursor, intermediates, and products will be used to identify cleavage sites by reference to the known structure of the precursor. Comparison of peptide maps of the precursor synthesized in a cell-free translation system to those from the precursor synthesized by intact cells will identify the region containing the "signal" sequence. Labelling with a lipid-soluble photoactivated probe will yield information on regions of membrane association. Characterization of precursor and intermediate cleaving activity in bag cell homogenates should reveal the degree of multiplicity of the processing endoproteases, and subcellular fractionation techniques will be used to determine their subcellular distribution. These experiments should yield insights into the fundamental cellular processes controlling the production of neuropeptides and peptide hormones.