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.
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.
