Purification and characterization of an actin-, calmodulin- and tropomyosin-binding protein from chicken gizzard smooth muscle.
An actin-binding protein (p33) has been purified from chicken gizzard smooth muscle. The homogenous protein has a molecular weight near 33000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. Its binding ability to F-actin remained after heating at 95 degrees C for 4 min. Immunoblot analyses indicated that p33 was not a degradation product from higher molecular components. The binding of p33 to F-actin was saturable in a molar ratio of about one p33 to 2-3 actin molecules with an apparent binding constant of 6.6 x 10(7) M-1. p33 also bound to calmodulin and tropomyosin. The bindings of p33 to F-actin and tropomyosin were regulated by calmodulin in a Ca(2+)-dependent fashion. In addition to actin, caldesmon and tropomyosin, p33 was contained in the native thin filaments prepared from smooth muscle. Other actin-binding proteins, including alpha-actinin, caldesmon and filamin, had little effect on p33 binding to actin filaments. These results demonstrate that p33 may function in actin-based cellular processes which are mediated by Ca2+ and calmodulin.
Fujii
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Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan.
D000818:Animals; D015153:Blotting, Western; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002645:Chickens; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000378:metabolism*; D008840:Microfilament Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism*; D051348:Tropomodulin
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D008840:Microfilament Proteins; D051348:Tropomodulin