The goal is to describe in as complete a manner as possible the chemical nature of force-generation in muscle. Specifically new photoaffinity analogues of ATP will be synthesized and used to label covalently active site residues of myosin from both straited (skeletal and cardiac) and non-straited (stomach and arterial) myosins. The subunit location and the amino acid composition of the labeled peptides will be determined by use of high performance liquid chromatography and gas phase sequencing techniques. The ATP analogues used in each case will be trapped at the active site eigter by chemical cross-linking two kinetically reactive thiols, SH1 and SH2, or by forming a stable transition state complex of ADP analogues with vanadate ions. Either of these two approaches allows the photoaffinity analogue myosin complex to be purified free of extraneous photolabels and greatly increases both the specificity and the extent of labeling. Preliminary results indicate that the subunit composition of the active site of smooth muscle myosin (stomach) is formed by elements of both the essential light chains and the heavy chains. Conversely, the active sites of straited muscle myosins appear to be composed only of heavy chains. This dichotomy will be investigated further utilizing new ATP analogues and myosins from other smooth muscles and from non-muscle tissues. A major goal will be to determine if there is a fundamental difference in the composition and nature of the force generating sites of smooth muscle myosins (and by analogy, non-muscle myosins) from those of straited muscles. This difference is not apparent from the gross morphology and subunit composition of the myosins from different tissues but may reflect the known differences in how contraction is regulated in these muscles. Other goals will be to synthesize new ATP analogues to be used (i) in affinity columns to purify myosin, (ii) as fluorescent probes of the heads of myosin and (iii) as electron-rich probes to aid in the solution of the X-ray structure of the recently crystallized head-regions of myosin. The long-term goal of this research is to provide a molecular explanation for how the chemical energy in ATP is converted into the mechanical energy of muscle contraction.
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