Abstract
Recent structural evidence (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65) suggests that the two heads of skeletal muscle myosin interact when the protein is bound to filamentous actin. Direct chemical cross-linking experiments show that the two heads of smooth muscle myosin interact in the presence of filamentous actin and the absence of ATP (Onishi, H., Maita, T., Matsuda, G., and Fujiwara, K. (1992) Biochemistry 31, 1201-1210). Head-head interactions may be important in the mechanism of phosphorylation-dependent regulation of smooth muscle myosin. To explore the structural elements essential for phosphorylation-dependent regulation, we purified a proteolytic fragment of chicken gizzard myosin containing only one head attached to an intact tail. This molecule contained a partially digested regulatory light chain, which was replaced with exogenously added intact light chain in either the thiophosphorylated or the unphosphorylated state. Control experiments showed that this replacement was nearly quantitative and did not alter the actin-activated ATPase of this myosin. Electron micrographs confirmed that the single-headed preparation contained an intact form of single-headed myosin. The unphosphorylated single-headed myosin hydrolyzed ATP rapidly and moved actin filaments in an in vitro motility assay. Phosphorylation had minimal effects upon these properties. Therefore, we conclude that phosphorylation-dependent regulation in this myosin requires two heads. These findings may have important implications in studies of other regulated motor proteins that contain two motor domains.
Highlights
THE JOURNAL OF BIOLOGICAL CHEMISTRYVol 270, No., Issue of February 3, pp. 2171-2175, 1995 Printed in U.S.A. Two Heads Are Required for Phosphorylation-dependent Regulation of Smooth Muscle Myosin*
Suggest that the "oft" state requires head-head or head-tail interactions. To differentiate between these two possibilities, we have investigated a highly purified single-headed proteolytic fragment isolated from a proteolytic digest of smooth muscle myosin
In our studies of proteolytic digestion of smooth muscle myosin, we found that the single-headed form is a transient intermediate that is never present in large amounts relative to the total number of myosin heads in a mixture
Summary
Vol 270, No., Issue of February 3, pp. 2171-2175, 1995 Printed in U.S.A. Two Heads Are Required for Phosphorylation-dependent Regulation of Smooth Muscle Myosin*. Similar experiments with unpurified smooth muscle myosin digests suggest that the single-headed myosin lacks phosphorylation-dependent regulation [15] This result contradicted an earlier report of the insoluble fraction of papain digests of smooth muscle myosin, suggesting that the single-headed form required phosphorylation for actin-activated ATPase [16]. From these initial studies with unpurified single-headed fragments, it is not clear whether the structural interactions responsible for the two myosin-linked regulatory mechanisms, Ca2 + binding and phosphorylation, are fundamentally different or whether they might be similar. Isolation of a single-headed fragment in a purified form has allowed an investigation of the phosphorylation-dependent regulatory behavior by steady-state ATPase and in vitro motility measurements
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