Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding.
Small bipolar filaments, or "minifilaments," are formed when smooth muscle myosin is dialyzed against low ionic strength pyrophosphate or citrate/Tris buffers. Unlike synthetic filaments formed at approximately physiological ionic conditions, minifilaments are homogeneous as indicated by their hypersharp boundary during sedimentation velocity. Electron microscopy and hydrodynamic techniques were used to show that 20-22S smooth muscle myosin minifilaments are 380 nm long and composed of 12-14 molecules. By varying solvents, a continuum of different size polymers in the range of 15-30S could be obtained. Skeletal muscle myosin, in contrast, preferentially forms a stable 32S minifilament (Reisler, E., P. Cheung, and N. Borochov. 1986. Biophys. J. 49:335-342), suggesting underlying differences in the assembly properties of the two myosins. Addition of salt to the smooth muscle myosin minifilaments caused unidirectional growth into a longer "side-polar" type of filament, whereas bipolar filaments were consistently formed by skeletal muscle myosin. As with synthetic filaments, addition of 1 mM MgATP caused dephosphorylated minifilaments to dissociate to a mixture of folded monomers and dimers. Phosphorylation of the regulatory light chain prevented disassembly by nucleotide, even though it had no detectable effect on the structure of the minifilament. These results suggest that differences in filament stability as a result of phosphorylation are due largely to conformational changes occurring in the myosin head, and are not due to differences in filament packing.
Trybus
K M
KM
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.
Lowey
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D000818:Animals; D002021:Buffers; D002417:Cattle; D002951:Citrates; D011756:Diphosphates; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000648:ultrastructure*; D009131:Muscle, Smooth, Vascular / Q000648:ultrastructure; D009218:Myosins / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D014461:Ultracentrifugation
D002021:Buffers; D002951:Citrates; D011756:Diphosphates; D010750:Phosphoproteins; D009218:Myosins