Extraction and reconstitution of calponin and consequent contractile ability in permeabilized smooth muscle fibers.
We demonstrate reduction and restoration of contractile ability in response to protein extraction and reconstitution in Triton X-100/glycerol-permeabilized smooth muscle fibers. Through significant reduction in the content of caldesmon (CaD), calponin (CaP), and the 20-kDa regulatory light chain (RLC) of myosin, but not other contractile proteins in "chemically skinned" fibers, we substantially reduced the contractile ability of these fibers, as measured by their ability to generate isometric force and to hydrolyze ATP by actomyosin Mg2+ ATPase. When the protein-depleted fibers were then reconstituted (either with a mixture of purified protein standards of CaD, CaP, and myosin RLC or with a protein extract from the demembranized muscle fibers containing CaD, CaP, and myosin RLC plus several low-molecular-mass proteins), all proteins used for reincorporation returned nearly to control levels, as did isometric force generation and rate of ATP hydrolysis. The fact that the low-molecular-mass proteins do not affect contractility in this model system indicates that our methods for reversible modulation of the content of CaP and CaD may provide a valuable tool for studying the thin-filament-based regulation of contractility.
Szymanski
Pawel T
PT
Physiology Program, Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA. pszymans@hsph.harvard.edu
Dickie
Renee
R
Rogers
Rick
R
Fredberg
Jeffrey J
JJ
D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D001696:Biomechanical Phenomena; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002463:Cell Membrane Permeability; D004285:Dogs; D008274:Magnesium / Q000378:metabolism; D008840:Microfilament Proteins; D009119:Muscle Contraction; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000502:physiology*; D009130:Muscle, Smooth / Q000737:chemistry / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D013997:Time Factors; D000096985:Calponins
D000205:Actomyosin; D002118:Calcium; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008274:Magnesium; D008840:Microfilament Proteins; D009218:Myosins; D000096985:Calponins