BIOELASTIC/ACOUSTIC SENSORS FOR SENSITIVE KINASE ASSAYS

磷酸化(通过激酶)和去磷酸化(通过磷酸酶) 代表了可能是最重要的中心酶过程 细胞功能和功能障碍。许多疾病状态，包括 癌症和阿尔茨海默病的特点是异常 磷酸化。这些酶活性的测定通常使用 放射性同位素磷-32，一种高能的贝塔辐射体。这一阶段 I SBIR建议开发一种现成的、敏感的和选择性的生物弹性材料- 肌动蛋白和磷酸酶的声学分析是对一种MGMS的反应 征集(SBIR 96-2)《减少对新酶测定的依赖》 放射性同位素。“ 生物弹性材料，弹性蛋白质聚合物，包括 重复的多肽序列，表现出相变行为， 在交联型弹性矩阵中被视为大于100% An的溶胀/消胀变化和酶促磷酸化 掺入特定的激酶位点是单一的最敏感的手段 推动弹性矩阵的膨胀。声学传感器检测 尺寸变化1%量级，有潜力实现 检测到0.1%的维度变化。这项建议是为了开发一种 一种灵敏、选择性、简便的检测肌酶活性的方法 在粗组织匀浆或提取物中，甚至在组织本身中 朝向设计的生物弹性基质内的特定多肽序列。 具体目标是：1)设计六种弹性蛋白质聚合物， 三个包含一个由蛋白激酶C唯一磷酸化的位点和 三个包含环AMP依赖的蛋白激酶位点，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) 模型蛋白质中溶胀/消胀转变的特征 及其交联型弹性矩阵，4)酶促 使模型蛋白质和它们的弹性基质磷酸化 描述对过渡的影响，以及5)利用声学 传感器检测包含的激酶位置的尺寸变化 由激酶和磷酸酶的活性产生的弹性基质。 拟议的商业应用：磷酸化和去磷酸化 组织中的活动是许多疾病状态的核心，包括 许多癌症和阿尔茨海默氏症。一种新型的选择性和可控性 用于检测这些活性的敏感传感器，而不是通常使用的 放射性同位素，但具有在原始组织上起作用的能力 提取物，甚至活组织上的提取物都将具有重大的商业价值。

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.