IN VIVO MEASUREMENTS OF ENZYME CHANNELING

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定是研究者机构。许多代谢途径中的中间产物从一个途径酶“引导”到下一个途径酶的假设被广泛接受，但不是普遍接受。它仍然存在争议的一个原因是许多弱酶复合物在分离过程中解离。通道效应可能适用的一个原因是，细胞中没有足够的水来支持KM处所有途径中间体的均匀浓度，传统上假定的允许途径发挥作用所需的近似浓度。Srere假设存在“代谢子”，即与稳定复合物相对的途径酶的瞬时缔合（例如电子传递链的细胞色素复合物或色氨酸合酶亚基的共价键）。据推测，连续酶的接近性将导致第一种酶的产物在与本体培养基内相同种类的分子相比时在竞争第二种酶的活性位点方面具有优势。也就是说，途径中间体不是细胞内相同分子的同一池的一部分。在该途径中产生的中间体被“引导”到下一个酶。为了测试通道的前景，我们正在实施电喷雾质谱法结合离线离子交换色谱法来测量葡萄糖/半乳糖代谢过程中的产物磷酸糖的相对丰度。使用同位素标记的起始材料，我们将能够看到含有同位素的产品是否用未标记的材料稀释（无通道）或保持未稀释（通道）。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The hypothesis that intermediates in many metabolic pathways are "channeled" from one pathway enzyme to the next is widely, but not universally, accepted. One reason it remains controversial is that many of the weak enzyme complexes are dissociated during isolation. One reason that channeling may apply is that there is not enough water in the cell to support uniform concentrations of all pathway intermediates at KM, the approximate concentration traditionally assumed required to permit pathways to function. Srere postulated the existence of "metabolons," transient associations of pathway enzymes as opposed to stable complexes (e.g. cytochrome complexes of the electron transport chain or the covalent linkage of tryptophan synthase subunits). It is postulated that the proximity of sequential enzymes would cause the product of the first enzyme to have an advantage in competition for the active site of the second enzyme when compared to molecules of the same species within the bulk medium. That is, pathway intermediates are not part of the same pool as are identical molecules within the cell. Intermediates produced within the pathway are "channeled" to the next enzyme. To test the prospect of channeling, we are implementing an electrospray mass spectrometry method coupled with off-line ion exchange chromatography to measure the relative abundances of sugar phosphates that are products in the glucose/galactose metabolism process. Using isotope labeled starting materials, we will be able to see whether the products containing the isotopes are diluted with unlabeled materials (no channeling) or remain undiluted (channeling).