LOCALIZATION OF SMOOTHELIN IN AVIAN SMOOTH-MUSCLE AND IDENTIFICATION OF A VASCULAR-SPECIFIC ISOFORM

Smoothelin is a smooth muscle-specific protein of minor abundance firs t identified via a monoclonal antibody obtained using an avian gizzard extract as antigen. Dual labelling of ultrathin sections with antibod ies to smoothelin together with antibodies to other smooth muscle prot eins showed that smoothelin was co-distributed with filamin and desmin in the cytoskeleton domain of the smooth muscle cell. From the findin g that smoothelin, unlike desmin, was readily extracted by Triton X-10 0 as well as under conditions that solubilized myosin, beta-actin and filamin, we conclude that smoothelin is most likely associated with th e actin cytoskeleton. Western blot analysis of gizzard smooth muscle t issue revealed an immunoreactive protein band with an apparent molecul ar weight of 59 kDa that separated into 3-4 isolated variants, while a vian vascular muscle showed a polypeptide band of 95 kDa. These result s point to the presence of specific isoforms in visceral and vascular smooth muscles. The 59 kDa isoform was shown to be distinct from the 6 0 kDa filamin-binding protein, described by Maekawa and Sakai (FEBS Le tt. 221, 68-72, 1987). As compared to other smooth muscle markers, suc h as calponin and SM22, smoothelin appeared very late during different iation in the chick gizzard, on about the 18th embryonic day. (C) 1997 Federation of European Biochemical Societies.