Elucidation of physiologically relevant protein-protein interactions through fluorescence spectroscopy and allied biophysical methods is the continuing theme of our research. The proteins and peptides involved in this proposal generally have functions in muscle contraction and/or cellular motility. Structure-function relationships in calmodulin will be investigated through fluorescence time decay measurements on the native protein and amino acid sequencing studies of dansylcalmodulin, an unusually responsive covalent conjugate. A search of various tissues will be made for small peptide calmodulin antagonists which may function intracellularly in the regulation of calmodulin-dependent enzymes. Any peptides isolated will be analyzed by gas phase amino acid sequencing. Their effectiveness and specificities as enzyme inhibitors will be demonstrated in catalytic assays of smooth muscle myosin light chain kinase, calcineurin, and the cAMP-dependent protein kinase. We will develop a fluorescent substrate to use in stopped flow kinetic studies of the catalytic mechanism of myosin light chain kinase. The in vitro association of phosphofructokinase with cytoskeletal proteins--actin and tubulin--will be examined in steady state fluorescence anisotropy measurements on covalent conjugates and in catalytic activity determinations with the unmodified proteins. Two new proteins from smooth muscle will be characterized in terms of molecular weight and amino acid composition. Their apparent structural role in smooth muscle will be investigated through fluorescence binding studies performed in vitro with myofibrillar proteins. This information will contribute generally to understanding of cellular motility and glycolysis. The small peptide studies may provide new clues on the physiological functions of polypeptide hormones. The effects of calmodulin on the mast cell degranulation and histamine release stimulated by the venoms of honey bees and other insects may suggest the design of new therapeutic agents. Other methods used: affinity chromatography, electrophoresis, analytical ultracentrifugation, circular dichroism spectroscopy.
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