BIOLOGY OF HYALURONIDASES

透明质酸酶是一组中性和酸性的酶 在整个动物界，对诸如 细胞运动、胚胎发育、伤口愈合、血管生成和 肿瘤发生学。尽管这些酶明显无能为力，但它们并不 由于缺乏一种快速、灵敏的检测方法，人们对此非常了解。这 现在我们开发了一种类似于ELISA的检测方法，这个问题已经被克服了 足够灵敏，以便可以执行动力学，并且活动 在小样本中可以测量。在一项器官调查中，高水平的 肝、肺、肾、脾中均有活性。肝酶 以及血清中的酶现在已经被提纯到均一 确实是不同的蛋白质。透明质酸酶似乎是一个广泛的家族 酶蛋白的含量。我们建议分离这个家族的其他酶， 并使用协调的生化、免疫学和分子生物学 方法，检查每种形式的生物学，识别前驱形式，以及 在培养细胞和组织中建立精确的组织定位 横断面。生物素标记的HA结合蛋白探针也将允许 底物的同时可视化。我们预测淋巴管 以脾酶为代表的系统是分解代谢的主要途径。 供房委会使用。肝脏和肾脏负责额外的 循环中游离HA的分解代谢途径，可能是 层次取决于HA聚合物的大小。成纤维细胞中的透明质酸酶 现在，通过直接化验和利用Western印迹也可以检测到 多克隆抗体针对纯化的肝酶。 检测这种中性酶的活性，以前没有观察到 正常的哺乳动物成纤维细胞，通过提取 高盐的存在，这似乎分解了一种酶抑制剂 很复杂。我们将使用适当的cDNA文库进行测序 透明质酸酶。共有序列以及模块结构域 将在这个新的酶家族中确定模式。为什么会有这样的情况 脊椎动物组织中广泛存在的透明质酸酶？为什么 HA底物本身存在于如此广泛的各种状态中：在 细胞结合的、完全水合的或与光谱有关的循环 透明质酸结合蛋白。为了找到这些问题的答案，我们将 试图将酶动力学与透明质酸酶的酶型联系起来 蛋白质在不同组织中的结构与分子的状态和位置 随之而来的透明质酸底物和对组织特异性的要求 功能。

Hyaluronidases are a group of neutral- and acid- active enzymes found throughout the animal kingdom, critical to such fundamental process as cell motility, embryological development, wound healing, angiogenesis and tumorigenesis. Despite their obvious impotence, these enzymes are not well understood because of the lack of a rapid, sensitive assay. This problem has now been overcome with our development of an ELISA-like assay sufficiently sensitive so that kinetics can be performed, and activity in small samples can be measured. In an organ survey, high levels of activity were found in liver, lung, kidney and spleen. The liver enzyme as well as the serum enzyme have now been purified to homogeneity an dare indeed different proteins. Hyaluronidases appear to be a broad family of enzyme proteins. We propose to isolate other enzymes of this family, and using a coordinated biochemical, immunological, and molecular biology approach, examine the biology of each, identify precursors forms, and establish precise histolocalization, both in cultured cells and in tissue sections. The biotinylated HA-binding protein probe will also permit simultaneous visualization of substrate. We predict that the lymphatic system, as exemplified by the spleen enzyme, is the major catabolic route for HA. The liver and the kidneys are responsible for additional catabolic pathways, of free HA in the circulation in what may be a hierarchy dependent on HA polymer size. The hyaluronidase in fibroblasts has also been detected now, by direct assay and by Western blot utilizing the polyclonal antibodies directed against the purified liver enzyme. detection of this neutral enzyme activity, not observed previously in normal mammalian fibroblasts, was facilitated by extracting in the presence of high salt, which appears to disaggregate an enzyme-inhibitor complex. We will use appropriate cDNA libraries to sequence hyaluronidase enzymes. Consensus sequences, as well as modular domain patterns will be identified in this new enzyme family. Why is there such a wide repertoir of hyaluronidase in vertebrate tissue? And why does the HA substrate itself exist in such a wide variety of states: free in the circulation, cell-bound, fully hydrated or in association with a spectrum of HA-binding proteins. Toward an answer to such questions, we will attempt to correlate enzyme kinetics and patterns of hyaluronidase protein structure in various tissues with the molecular state an location of the attendant HA substrate and the requirements for tissue-specific functions.