Identification of a 115-kDa protein from muscle tissues: expression of a novel nonmuscle alpha-actinin in vascular endothelial cells.
A novel nonmuscle alpha-actinin from chicken lung tissue is distinct from reported nonmuscle and muscle alpha-actinins in its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 6 M urea (apparent molecular mass is 115 kDa) and its Ca(2+)-sensitivity in actin binding (M. Imamura and T. Masaki, J. Biol. Chem. 267, 25927-25933, 1992). SDS-PAGE analysis of extract from chicken gizzard smooth muscle tissue in the presence of urea showed the existence of a protein migrating at 115 kDa. Analyses of proteolytic digestion and partial amino acid sequence revealed that the latter 115-kDa protein is identical or very similar to the Ca(2+)-insensitive lung alpha-actinin. We prepared an antibody specific for the 115-kDa protein by affinity column coupled with the purified lung alpha-actinin, and the expression and distribution of the 115-kDa protein in muscle tissues were examined. Immunoblotting and immunofluorescence microscopy demonstrated that the 115-kDa protein was expressed in heart and pectoralis muscle tissues as well as in gizzard. Double-fluorescence staining with the anti-115-kDa protein antibody and anti-muscle alpha-actinin antibody indicated that the 115-kDa protein was localized in tubular structures along the axes of muscle fibers, whose diameter was 3.5-5 microns. The tubular structure did not respond to the muscle cells. Vascular-labeling analysis with carbon dye showed that the staining with anti-115-kDa protein antibody was located along the inside of the vascular wall. These results indicated that the Ca(2+)-insensitive alpha-actinin was expressed in vascular endothelial cells and suggested that its Ca(2+)-sensitivity was related to the function of the endothelial cells.
Imamura
M
M
Department of Pharmacology, University of Tsukuba, Ibaraki, Japan.
Masaki
T
T
D000185:Actinin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002645:Chickens; D004730:Endothelium, Vascular / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D008168:Lung / Q000378:metabolism; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D011817:Rabbits; D014018:Tissue Distribution
D000185:Actinin; D002118:Calcium