Characterization of an inhibitor of actin polymerization in vinculin-rich fraction of turkey gizzard smooth muscle.
We report here on the purification and characterization of a new 25-kDa inhibitor of actin polymerization from turkey gizzard smooth muscle. The protein was purified by chromatography on DEAE-cellulose and hydroxyapatite, as well as by affinity chromatography on an immobilized-antibody column. The purified polypeptide reduced the low-shear viscosity of actin, apparently due to its inhibitory effect on actin polymerization. We demonstrate that this protein is largely responsible for the apparent inhibitory activity previously reported to be associated with smooth muscle vinculin preparations. Three independent monoclonal antibodies prepared against the 25-kDa inhibitor of actin polymerization can effectively adsorb the inhibiting activity of actin polymerization from the crude vinculin preparation or inhibit it. We also show here that the 25-kDa inhibitor of actin polymerization tends to undergo dimerization when maintained in non-reducing buffers, concomitant with the loss of its inhibitory activity. Immunohistochemical labeling of frozen sections, as well as immunoblotting analyzes, indicated that the 25-kDa inhibitor of actin polymerization is particularly enriched in smooth muscle cells and that its distribution is apparently homogenous throughout the cytoplasm showing no apparent enrichment in the vinculin-rich dense plaques located along the endofacial surface of the plasma membrane.
Miron
T
T
Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Wilchek
M
M
Geiger
B
B
D000199:Actins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001704:Biopolymers / Q000378:metabolism; D002645:Chickens; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005895:Gizzard, Avian / Q000378:metabolism*; D007700:Kinetics; D008854:Microscopy, Electron; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D014422:Turkeys; D016596:Vinculin
D000199:Actins; D001704:Biopolymers; D009124:Muscle Proteins; D016596:Vinculin