BIOELASTIC/ACOUSTIC SENSORS FOR SENSITIVE KINASE ASSAYS

磷酸化（通过激酶）和去磷酸化（通过磷酸酶） 可能代表了最重要的酶促过程， 细胞功能和功能障碍。多种疾病，包括 癌症和阿尔茨海默病的特征是异常的 磷酸化这些酶活性的测定通常利用 放射性同位素磷-32一种高能β发射体 这个阶段 I SBIR建议开发一种现成的，敏感的和选择性的生物弹性- 激酶和磷酸酶的声学测定响应于MGMS 征求（SBIR 96-2）“新酶测定法，以减少对 放射性同位素" 生物弹性材料，弹性蛋白质基聚合物，包括 重复的肽序列，表现出相变行为， 在交联的弹性基质中， 溶胀/去溶胀变化，和酶促磷酸化， 掺入的特异性激酶位点是唯一最敏感的方法， 驱动弹性基质的膨胀。声学传感器检测到 1%量级的尺寸变化， 检测到0.1%的尺寸变化。该提案旨在制定一项 用于检测激酶活性的灵敏、选择性、简便的测定 在粗组织匀浆或提取物中或甚至在组织本身中 在设计的生物弹性基质内的特定肽序列。 具体目标是：1）设计六种弹性蛋白质基聚合物， 三个含有被蛋白激酶C唯一磷酸化的位点， 三个含有环AMP依赖性蛋白激酶位点，2）至 利用分子生物学技术制备模型蛋白， 基因的构建和表达，发酵和纯化，3） 表征模型蛋白质中的溶胀/去溶胀转变 和它们的交联弹性基质，4）酶促地 磷酸化模型蛋白及其弹性基质， 表征对过渡的影响，以及5）利用声学 传感器检测含激酶位点的 由激酶和磷酸酶的活性产生的弹性基质。 拟定商业应用：磷酸化和去磷酸化 组织中的活性是许多疾病状态的核心，包括 许多癌症和老年痴呆症。一种新型的选择性和 灵敏的传感器来测定这些活性，而不需要通常使用 放射性同位素，但有能力对天然组织起作用， 提取物或甚至在活组织上将具有显著的商业价值。

Phosphorylation (by kinases) and dephosphorylation (by phosphatases) represent perhaps the central most important enzymatic processes of cellular function and dysfunction. Numerous disease states, including cancers and Alzheimer's disease, are characterized by aberrant phosphorylation. The assays for these enzyme activities normally utilize the radio-isotope, phosphorus-32, a high energy beta-emitter. This Phase I SBIR proposal to develop a ready, sensitive and selective bioelastic- acoustic assay for kinases and phosphatases is in response to an MGMS solicitation (SBIR 96-2) for "New enzyme assays to reduce reliance on radioisotopes." Bioelastic materials, elastic protein-based polymers comprised of repeating peptide sequences, exhibit phase transitional behavior which in the cross-linked elastic matrices is seen as greater than 100% swelling/de-swelling changes, and enzymatic phosphorylation of an incorporated specific kinase site is the single most sensitive means of driving swelling of the elastic matrices. Acoustical sensors detect dimension changes of the order of 1% with the potential to achieve detection of 0.1% dimensional changes. This proposal is to develop a sensitive, selective, facile assay for the detection of kinase activities in crude tissue homogenates or extracts or even in the tissue itself toward specific peptide sequences within a designed bioelastic matrix. The specific aims are: 1) to design six elastic protein-based polymers, three containing a site uniquely phosphorylated by protein kinase C and three containing a cyclic AMP dependent protein kinase site, 2) to prepare the model proteins utilizing the molecular biology techniques of gene construction and expression, fermentation and purification, 3) to characterize the swelling/de-swelling transition in the model proteins and their cross-linked elastic matrices, 4) to enzymatically phosphorylate the model proteins and their elastic matrices and characterize the effect on the transition, and 5) to utilize an acoustic sensor to detect the dimensional changes in the kinase site-containing elastic matrices resulting from the activity of kinases and phosphatases. PROPOSED COMMERCIAL APPLICATION: Phosphorylation and dephosphorylation activities in tissues are central to numerous disease states, including many cancers and Alzheimer's disease. A new type of selective and sensitive sensor to assay for these activities without the common use of radio-isotopes but with the capacity to be functional on crude tissue extracts or even on live tissue would have significant commercial value.