Smooth muscle caldesmon. Rapid purification and F-actin cross-linking properties.
A method for the rapid purification of caldesmon, an F-actin binding protein of smooth muscle, has been developed. Caldesmon remains native after heating at 90 degrees C, a property that provides the basis for the purification in high yield of both caldesmon and tropomyosin, another heat-stable protein of smooth muscle. Caldesmon purified by this procedure is a highly asymmetric protein with a sedimentation coefficient of approximately 2.7 S and a Stokes radius of about 91 A. The protein exists as two polypeptide chains of Mr = 135,000 and 140,000, with each Mr polypeptide being resolvable into several isoelectric species. Estimates based on densitometry of stained gels suggest that caldesmon is more abundant in smooth muscle than filamin or alpha-actinin. Purified caldesmon bound to F-actin in the pH range 6-8. Binding was unaffected by Ca2+ or Mg2+ at up to millimolar levels. Binding was saturable, with a polypeptide molar ratio of about one caldesmon to six actins at saturation. F-actin binding was not inhibited by saturating levels of tropomyosin. Caldesmon dramatically increased the viscosity of F-actin. Light microscopy and electron microscopy of negatively stained material revealed that caldesmon induced the formation of massive F-actin bundles which contained up to hundreds of filaments. Electron microscopy of sectioned caldesmon-saturated F-actin mixtures revealed large bundles which appeared to include linear arrays of regularly spaced actin filaments cut transversely, exhibiting a center to center spacing of 15 nm. Possible structural implications based on the existence of these structures is presented.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D002148:Calmodulin-Binding Proteins; D002645:Chickens; D005895:Gizzard, Avian / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D014335:Tropomyosin / Q000302:isolation & purification
D000199:Actins; D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D046911:Macromolecular Substances; D014335:Tropomyosin; D010749:Phosphoprotein Phosphatases