We have been studying the regulation of both skeletal and smooth muscle. In skeletal muscle we did this by examining the calcium sensitivity of weakly binding crossbridges. Since it was very difficult to do this with the normally occurring weakly-binding myosin. ATP crossbridge, we instead found a way of creating ATPinsensitive weakly-binding crossbridges. This was accomplished by treating fibers with either para-phenylenedimaleimide (pPDM) or with N-phenyl maleimide (NPM). We found that these pPDM- or NPM- crossbridges, in the presence or absence of ATP, had binding strength and attachment and detachment rate constants virtually identical to normal myosin.ATP crossbridges. Unlike with myosin.ATP crossbridges, we were able to examine the calcium sensitivity of these artificially created crossbridges without concern about the effects of ATP hydrolysis. As postulated for myosin.ATP crossbridges based upon solution studies, we found that the binding strength and rate constants of the pPDM- and NPM-crossbridges in fibers are relatively insensitive to changes in calcium over the physiological range. In smooth muscle we examined the molecular mechanism of action of two compounds which stimulate adenylate cyclase and are known to relax smooth muscle, vasoactive intestinal peptide (VIP) and isoproterenol. Results to date show that VIP, when it causes relaxation, also causes a reduction in the percentage phosphorylation of the 20 kdalton smooth muscle myosin light chain. In vitro, phosphorylation of myosin light chain kinase, the enzyme that phosphorylates the myosin light chains, reduces its activity. We are currently examining whether a similar mechanism of regulation might function in fibers. To date we have labeled the ATP pool of small samples of smooth muscle, isolated the kinase through immunoprecipitation, and have found that samples relaxed with VIP have increased phosphate incorporation. We are currently examining the site of this increased phosphorylation to see if it is the same as the site phosphorylated in vitro.
