Identification by monoclonal antibodies and characterization of human platelet caldesmon.
Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross-reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin-Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001792:Blood Platelets / Q000032:analysis*; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000276:immunology*; D002352:Carrier Proteins / Q000276:immunology*; D002645:Chickens; D005455:Fluorescent Antibody Technique; D005782:Gels; D018260:Gelsolin; D005895:Gizzard, Avian / Q000032:analysis; D006801:Humans; D008840:Microfilament Proteins / Q000276:immunology*; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D005782:Gels; D018260:Gelsolin; D008840:Microfilament Proteins; C016953:brevin