title: Effects of phosphorylation by myosin light chain kinase on the structure of Limulus thick filaments.
abstract: The results discussed in the preceding paper (Levine, R. J. C., J. L. Woodhead, and H. A. King. 1991. J. Cell Biol. 113:563-572.) indicate that A-band shortening in Limulus muscle is a thick filament response to activation that occurs largely by fragmentation of filament ends. To assess the effect of biochemical changes directly associated with activation on the length and structure of thick filaments from Limulus telson muscle, a dually regulated tissue (Lehman, W., J. Kendrick-Jones, and A. G. Szent Gyorgyi. 1973. Cold Spring Harbor Symp. Quant. Biol. 37:319-330.) we have examined the thick filament response to phosphorylation of myosin regulatory light chains. In agreement with the previous work of J. Sellers (1981. J. Biol. Chem. 256:9274-9278), Limulus myosin, incubated with partially purified chicken gizzard myosin light chain kinase (MLCK) and [gamma 32P]-ATP, binds 2 mol phosphate/mole protein. On autoradiographs of SDS-PAGE, the label is restricted to the two regulatory light chains, LC1 and LC2. Incubation of long (greater than or equal to 4.0 microns) thick filaments, separated from Limulus telson muscle under relaxing conditions, with either intact MLCK in the presence of Ca2+ and calmodulin, or Ca2(+)-independent MLCK obtained by brief chymotryptic digestion (Walsh, M. P., R. Dabrowska, S. Hinkins, and D. J. Hartshorne. 1982. Biochemistry. 21:1919-1925), causes significant changes in their structure. These include: disordering of the helical surface arrangement of myosin heads as they move away from the filament backbone; the presence of distal bends and breaks, with loss of some surface myosin molecules, in each polar filament half; and the production of shorter filaments and end-fragments. The latter structures are similar to those produced by Ca2(+)-activation of skinned fibers (Levine, R. J. C., J. L. Woodhead, and H. A. King. J. Cell Biol. 113:563-572). Rinsing experimental filament preparations with relaxing solution before staining restores some degree of order of the helical surface array, but not filament length. We propose that outward movement of myosin heads and thick filament shortening in Limulus muscle are responses to activation that are dependent on phosphorylation of regulatory myosin light chains. Filament shortening may be due, in large part, to breakage at the filament ends.
authors.affiliation: Department of Anatomy and Neurobiology, Medical College of Pennsylvania, Philadelphia 19129.
authors.forename: R J
authors.initials: RJ
authors.lastname: Levine
authors.forename: P D
authors.initials: PD
authors.lastname: Chantler
authors.forename: R W
authors.initials: RW
authors.lastname: Kensler
authors.forename: J L
authors.initials: JL
authors.lastname: Woodhead
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D005583:Fourier Analysis; D006737:Horseshoe Crabs; D008854:Microscopy, Electron; D009126:Muscle Relaxation; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium