The Chemical Core has the long term objective and specific aim of providing a unique resource of fluorescent and photosensitive """"""""caged"""""""" nucleotides for exploring mechanisms of smooth muscle physiology and cell biology. The specific health benefits of the research relevant to this program project are set out in the Introduction (Significance"""""""" and in the individual projects. The Core will achieve its goals principally through (1) synthesis of coumarin-labeled nucleotides that will enable crossbridge states to be identified and their rates of formation and decay measured; (2) the synthesis of novel caged nucleotides and regulators of smooth muscle including caged phosphorylated myosin regulatory light chains. The methods for achieving these goals will be through chemical protocols established in the laboratories of the principal investigators. This synthetic chemistry program will be supported by rigorous characterization of the probes (by monitoring their activity and function when interacting with isolated muscle proteins) and the caged compounds (by monitoring their photochemical properties and (desirable) biological inertness). Further goals will be achieved by development of real-time probes of Pi and ADP that will be applied (Project 1) to record smooth muscle ATPase activity in various physiological states and pseudo- ATPase (myosin light chain kinase/smooth muscle phosphatase) activity. Finally, the reagents will, insofar as is practical, be tested in a physiological environment by probing their behavior in skinned skeletal muscle fibers. The expertise and collaboration of chemists, enzymologists and physiologists in the Division of Physical Biochemistry at NIMR make this a unique chemical core facility.
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